MARTHA ZARAIN-HERZBERG NORMA HERNANDEZ-SAAVEDRA FELIPE ASCENCIO-VALLE

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1 JOURNAL OF THE WORLD AQUACULTURE SOCIETY Vol. 34, No. 1 March, 2003 Biological Characterization of a Less Virulent Taura Syndrome in Pacific White Shrimp Litopenaeus vannamei (Crustacea: Decapoda): Gross Signs, Histopathological Lesions, and Mortalities MARTHA ZARAIN-HERZBERG Centro de Ciencias de Sinaloa, Avenida de las Ame'ricas 2271 Norte. Culiacdn. Sinuloa, Mgxico. cp NORMA HERNANDEZ-SAAVEDRA FELIPE ASCENCIO-VALLE Centro de Investigacidn Bioldgica del Noroeste, La Paz, Buja California, M6xico Taura syndrome (TS) was first recognized in shrimp farms located near the mouth of the Taura River in the Gulf of Guayaquil, Ecuador in mid (JimCnez 1992; Lightner et al. 1995), where the disease caused catastrophic losses with cumulative mortality rates from 60% to >90% of affected pond-cultured, juvenile Pacific white shrimp Litopenaeus vannamei (Perez Farfante and Kensley 1997). The geographic distribution of the disease now includes Ecuador, the adjacent region of Peru, the Pacific and Caribbean coast of Colombia, Honduras, Guatemala, El Salvador, northeast Brazil, Nicaragua, Belize, the states of Hawaii, Florida, and Texas in the USA, the Mexican states of Sonora, Sinaloa, Chiapas, and Guerrero, and recently Taiwan (Lightner 1996; Chien et al. 1999). The epizootiological and laboratory studies that followed its discovery and spread from Ecuador showed that Taura syndrome (TS) had a viral etiology in the American (L. vannamei, L. stylirostris, L. schmitti, L. setiferus, Farfuntepenaeus aztecus) and Asian (Penaeus monodon, Marsupenaeus japonicus, Fenneropenaeus chinensis) penaeid species naturally or experimentally infected by the virus. Litopenaeus vannamei was by far the most severely affected (Brock et al. 1995, 1997; Hasson et al. 1995, 1999b; Lightner 1996; Overstreet et al. 1997). Infectivity studies demonstrated that TS could be induced in specific-pathogen-free (SPF) L. vannamei. Shrimp started dying after being fed TSV-infected tissue between day 4 or 5, with mortalities of 80% (Brock et al. 1995). Test animals were also infected by intramuscular injection with crude homogenate and cell-free suspensions, from TSV-infected Ecuatorian L. vannamei, causing mortalities of 73% to 87% among treated groups. All moribund shrimp demonstrated moderate to severe pathognomonic TS lesions (Hasson et al. 1995), which consist of pyknotic and karyorrhectic nuclei and generally spherical cytoplasmic inclusions with multifocal areas of necrosis of the cuticular epithelium giving TS lesions a peppered or buckshot-riddled appearance (Brock et al. 1995; Lightner et al. 1995; Hasson et al. 1995; Lightner 1999). The hemispheric geographic distribution of TS indicates that the same virus or a closely related one is responsible for the TS epizootics that have occurred throughout America since 1992 (Hasson et al. 1999a). Ecuatorian and Hawaiian TS virus isolates were found to be identical in their biophysical, biochemical, and biological characteristics, and they were considered to be the same virus (Bonami et al. 1997). In early 1995, TSV was detected in wild adult L. vannamei collected from the offshore fishery of the southern Mexican state of Chiapas, near its border with Guatemala and near where TS had first appeared in 8 Copyrtght by the World Aquaculture Society

2 I00 ZARAIN-HERZBERG ET AL. Guatemalan shrimp farms in 1994 (Lightner 1996; Zarain and Ascencio 2001). Losses in shrimp farms of Sinaloa, Mtxico, due to TS reached a peak in As a result of TS, shrimp production of Sinaloa decreased 37%, though in other countries the production had been reduced more severely (Lightner 1996). Since that time there has been a steady reduction in the number of shrimp farms infected, with stabilization in the production of shrimp by This stabilization was probably caused by the change in species cultivated from L. vannamei to L. stylirostris (Zarain and Ascencio 2001). In 1996 JimCnez (1996) reported a disease similar to Taura syndrome. The impact of this TS-like disease in Sinaloa ( ) suggests a diminishing virulence in the virus strain (TSVSIN98, Sinaloa, MCxico 1998 TSV) (Zarain and Ascencio 2001). Since the appearance of this strain, severity of losses have not been as great as those observed in other countries. Recently, TS has reemerged in species previously reported as refractory to TS. Recent evidence has shown a molecular difference between TSVSIN98 and other TSV strains (Erickson and Lightner 2001). Because of this, the objective of this study was to determine whether there are differences in the gross signs, histopathological lesions, and mortalities in L. vannamei due to challenge with the TSVSIN98 strain, compared to previous challenge studies with the typical TS virus. Materials and Methods Bioassay Experimental Shrimp Twelve 15-L aquaria, covered with glass, were each stocked with 15 healthy (free of TSV virus) L. vannamei (from CIBNOR, La Paz, Mexico) of 13.5 g average weight. Before beginning each bioassay, the shrimp were acclimated for 3 d. The experimental aquaria were located in an isolated room disinfected with calcium hypochlorite. Nets and other necessary tools were not exchanged between experimental tanks. A daily water exchange of 50% was made with prepared sea water (Instant Ocean Aquarium Systems, Mentor, Ohio, USA), maintaining a salinity of 30 ppt. Each tank was held at constant conditions: temperature maintained between 26 and 28 C, continuous aeration, and 10 h of light per day during the experiment. Shrimp were fed early in the morning and near noon, with commercial pellets (Malta Clayton, MCxico, 40% protein), on the basis of 5% body weight (Overstreet et al. 1997). Every day, the variables of water quality (ph, total ammonia-n, and nitrite) were recorded. Inoculum Preparation The inoculum was prepared from 100 g of macerated heads of TSV-infected L. vannamei originating from a single epizootic at a shrimp farm in Sinaloa during 1998 (TSVSIN98) and was homogenized in 300- ml Ringer s physiological saline (Hasson et al. 1995; Overstreet et al. 1997). This slurry was centrifuged for 15 min at 1,000 g. The supernatant was poured off and filtered through a No. 5 Whatman filter and a 0.45-km GF/A filter (Whatman International Ltd., Maidstone, England). This suspension was diluted and with sterile 2% saline, and these dilutions were used for the inoculations. The shrimp were injected with 0.10 ml (1% v/w) in the third abdominal segment (Overstreet et al. 1997). Sixty shrimp were injected with 2% saline for the control. The same number of organisms and replicates were used for the treatments with and dilution. The health of the shrimp in each aquarium was examined at least four times each day during 11 d post-challenge. At least one shrimp was taken daily for preservation, preferentially those, that showed some signs of disease or were moribund. The number of dead and moribund shrimp was also recorded.

3 CHARACTERIZATION OF A LESS VIRULENT TAURA SYNDROME 101 TABLE 1. Curnulative percentage niorralities per treatment and replicate and number of L. vannamei wirh TS lesions ut the end of 11 d of infectivity bioassay. Cumulative 76 mortalities Number of shrimp with TS lesions in Treatment-replicate on day 11 the four replicates and (day sampled) Control-Replicate 1 (0/15) 0.0 Control-Replicate 2 (0/15) 0.0 Control-Replicate 3 (0/15) 0.0 None Control-Replicate 4 (0/15) 0.0 Dilution Yl,,-Replicate I (8/15) 53.3 Dilution Yl,,-Replicate 2 (6/15) 40.0 Dilution '/,,,-Replicate 3 (7/15) (1), 2W, 2(3), 2(4), 1(5), I(@, Dilution '/,,,-Replicate 4 (5/15) 33.3 I@). Dilution I/l,,,-Replicate 1 (6/15) 40.0 Dilution I/l,,rReplicate 2 (6/15) 40.0 Dilution I/l,,,-Replicate 3 (7/15) (1), 2(2), 1(3), 2(4), 1(5), 1(6), Dilution I/,,,,-Replicate 4 (7/15) (7), 1(9). Histological and Hybridization Analysis The selected shrimp were injected and preserved with RF (RNA-friendly fixative) solution (Hasson et al. 1997). The histopathological methods used were routine procedures using hematoxylin and eosinphloxine (H&E) (Bell and Lightner 1988). The sections were examined for pathognomonic TS lesions (Lightner et al. 1995; Hasson et al. 1995). They were also analyzed using genetic probes by in situ hybridization (DiagXotics, Wilton, Connecticut, USA). At the end of the bioassay, the surviving organisms were frozen at -70 C. Results Gross Signs, Histopathology, and Hybridization The gross signs recorded for both dilutions were weakness, lethargy, abnormal swimming, expansion of red chromatophores over the entire body and appendages, necrosis of the cuticular epithelium in uropods, and soft cuticle. Shrimp with black spots were detected on day 9. Table 1 lists the day and number of shrimp that had characteristic acute phase or transition phase TSV lesions (as described by Hasson et al. 1999b) from day 1 until day 9 post-injection (Fig. la, B). The presence of chronic TSV lesions continued until the end of the experiment. The presence of hemocyte accumulations and the formation of spheroids in the lymphoid organ were observed beginning with the day 2 samples and persisting until the end of the experiment in both challenges dilutions and Sixty-six percent of the analyzed shrimp that had been challenged with the dilution had TSV lesions in the cuticular epithelium and in the lymphoid organ. These disease signs indicate that the shrimp were in the transition phase of the disease (Hasson et al. 1999b). The histological sections of shrimp displaying pathognomonic TSV lesions were subjected to a confirmatory test by in situ hybridization using specific TSV probes, with the results being positive in 90% of the assays. An intense reaction to the probes was observed in the cuticular epithelium in areas where necrosis was not evident and lightly positive within the lymphoid organ on the first day (Fig. 1C). Lesions continued to be observed through the last day of the experiment. Development of Mortalities Figure 2 shows the development of daily mortalities on each of the replicates using a dilution. Table 1 also shows the cumulative mortalities for each replicate of both dilutions as well as the controls at the end of the period. In both dilutions, we obtained

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5 CHARACTERIZATION OF A LESS VIRULENT TAURA SYNDROME 103 a cumulative mean mortality of 43%. Also, in both dilutions the maximum daily mortality was 10% (not shown). For shrimp challenged with the dilution, the mortalities began the first day post-injection, whereas for the shrimp challenged with the dilution, the mortalities did not begin until the second day post-injection. Discussion Taura syndrome is an infectious disease of viral etiology (Brock et al. 1995; Lightner et al. 1995; Hasson et al. 1995), which under experimental conditions can result in cumulative mortalities that range between 73% and 87%. Under culture conditions mortalities may reach 95% in L. vannamei. However, the effects of TS on the shrimp farming industry of Sinaloa, MCxico has not been as harmful as in other countries that cultivate this species. (Lightner 1996; Hasson et al. 1999a; Zarain and Ascencio 2001). TS virus (TSV) has appeared in different ambient conditions and with a variety of clinical manifestations. Although TSV is able to infect L. vannamei and L. stylirostris, the expression of the disease and the severity of its disease signs differ in each species. In general, L. stylirostris, F. duorarum, and F. aztecus are more tolerant to TSV than L. vannamei (Brock et al. 1995; Overstreet et al. 1997). The histological evaluation of moribund shrimp collected from this infectivity study between day 1 and 11 d post-injection showed pathognomonic TS lesions in the cuticular epithelium of appendages and car- FIGURE 2. L. vannamei daily mortality number disiribuiion in four replicates (Rep I. Rep 2, Rep 3, Rep 4) and one control in ihe infectivity bioassay, afrer induced infection by injection using inoculum tissue TSVSlN98 dilution over ihe I I d experiment. apace in general and the formation of spheroids in the lymphoid organ. Lesions were similar to those reported for organisms naturally or experimentally infected with viral isolates originating from Ecuador, Hawaii, and Texas (Brock et al. 1995; Lightner et al. 1995; Hasson et al. 1995, 1997, 1999b; Bonami et a1.1997). At the end of the 1 I-d bioassay, 66% of the shrimp inoculated by intramuscular in- jection with the diluted material from TSVSIN98 showed lesions characteristic of both the acute and transition phase of the disease. That the in situ hybridization test with the TSV-specific gene probe showed a strong positive reaction in areas of cuticular epithelium where no evidence of necrosis was apparent and with less intensity within the lymphoid organ suggests the virus was t FIGURE 1. Photomicrographs of hemutoxylin and eosin (HBE) hisiological seciions. (A) The abdomen of a juvenile L. vannamei. day I, diluiion. illusirating ihe characteristics of ihe TSV acute-phase lesion wiih cellular necrosis of the cuiicular epithelium, cytoplasmic inclusion bodies, and pyknotic and karyorrhectic nuclei (arrow). 400X magni3cation. (B) The cuticular and subcuiicular epidermis of a juvenile L. vannamei, day 2, dilution, illustrating the characteristic of TSV iransition phase. A melanized cuticule (arrowhead), necrosis wiihin cuticular epithelial cells, and hemocytic injiltraie are visible (arrow). IOOX magni3caiion. (C) Gene-probed histological section of cuticular epidermis of juvenile L. vannamei appendage, day 2. I//(, dilution, displuying an iniense posiiive reaction (black precipitate) (arrow). Sections were counter-stained with Bismarck bruwn. 400X magnijication.

6 104 ZARAIN-HERZBERG ET AL. already present within cells that have not yet undergone necrosis at the beginning of the infection. It is also possible that some viral particles may have been phagocytized by hemocytes, which in turn may have resulted in a positive reaction to the TSV gene probe within the lymphoid organ on the first day post-injection, and possibly causing the formation of spheroids from day 2 until the end of the experiment. It has been reported that during the normal course of a TSV episode some shrimp showing moderate to severe lesions may recover and survive (Lightner 1996; Hasson et al. 1999b). However our results suggest that a high percentage of the shrimp (L. vannarnei) used in this experiment may have been resistant to the infection or may have been only mildly affected with the disease, resulting in early recovery. That the first shrimp displaying lesions characteristic of the transition phase of TS disease was observed almost immediately after the first day post-injection and the average cumulative mortality only reached 43% seems to support this hypothesis. There is also the possibility that the apparent resistance or the mild pathogenicity of this strain of the virus could be caused by some genetic change experienced by the viral isolate employed, TSVSIN98 (Erickson and Lightner 2001), which could have decreased the potential to infect L. vannamei, hampering the ability to express the viral genome in the host. We speculate that the difference between the results obtained in this study and those previously reported in the literature could be caused by differences intrinsic to the environmental conditions from where the test shrimp originated and that may have influenced their susceptibility to infection. Additionally, since the TSV replication strategy does not include a repair system (Domingo and Holland 1997), there is a high probability for mutations that may have affected the ability of this strain of virus to infect the shrimp employed in this study. Further studies on the molecular character- ization of the TSVSLN98 are now in progress. (H.S. Erickson, University of Arizona, personal communication). Acknowledgments Funding for this research was provided by a grant from SIMAC (CONACyT) We thank Dr. Cistulo A. Alejo and Dr. Carlos Pantoja for their critical review and corrections to the manuscript. We thank Argelia Fueyo, Jorge Salcido, Celina Villegas, and Rosy Raygoza for their technical assistance. Special thanks to Dr. Donald Lightner for his critical reading and valuable comments. Thanks to Dr. Ellis Glazier for editing the English-language text. Literature Cited Bell, T. A. and D. V. Lightner A handbook of normal shrimp histology. Special publication No. 1. World Aquaculture Society, Baton Rouge, Louisiana, USA. Bonami, J. R., K. W. Hasson, J. Mari, B. T. Poulos, and D. V. Lightner Taura Syndrome of marine penaeid shrimp: characterization of the viral agent. Journal of General Virology 78: Brock, J. A., R. Gose, D. V. Lightner, and K. W. Hasson An overview on Taura syndrome, an important disease of farmed Penaeus vannamei. Pages in C. L. Browdy, J. and S. Hopkins, editors. Swimming through troubled water, Proceedings of the special session on shrimp farming. San Diego. California, USA. 1-4 February The World Aquaculture Society, Baton Rouge, USA. Brock, J. A., R. Gose, D. V. Lightner, and K. W. Hasson Recent developments and overview of Taura syndrome of fmed shrimp in America. Pages in T. W. Flegel and I. H. MacRae, editors. Disease in Asia aquaculture 111. Fish Health Section, Asian Fisheries Society, Manila, Philippines. Chien, T., H. Hsu-Tien, C. Sheng-Hsiung, H. Jung- Ping, K. Shu-Ting, L. Nan-Jung, H. Tien-Lai, L. Ming-Chang, and L. Shih-Yuh Taura syndrome in Pacific white shrimp Penaeus vunnamei cultured in Taiwan. Diseases of Aquatic Organisms 38: Domingo, E. and J. J. Holland RNA virus mutations and fitness for survival. Annual Reviews of Microbiology 51: Erickson, H. and D. V. Lightner Investigations into Taura syndrome virus (TSV) geographic

7 CHARACTERIZATION OF A LESS VIRULENT TAURA SYNDROME 105 and year isolate strain differences (abstract). Page 235 in World Aquaculture Society, editor. The annual meeting of the World Aquaculture Society, Orlando, Florida, USA January The World Aquaculture Society, Baton Rouge, Lousiana, USA. Hasson, K. W., J. Hasson, H. Aubert, R. M. Redman, and D. V. Lightner A new RNAfriendly fixative for the penaeid shrimp samples for virological detection using cdna genomic probes. Journal of Virology Methods 66: Hasson, K. W., D. V. Lightner, J. Mari, J. R. Bonami, B. T. Poulos, L. L. Mohney, R. M. Redman, and J. A. Brock. 1999a. The geographic distribution of Taura Syndrome Virus (TSV) in the Americas: determination by histopathology and in siru hybridization using TSV-specific cdna probes. Aquaculture 171: Hasson, K. W., D. V. Lightner, B. T. Poulos, R. M. Redman, B. M. White, J. A. Brock, and J. R. Bonami Taura syndrome in Penaeus vanname;: demonstration of viral etiology. Disease Aquatic Organisms 23: Hasson, K. W., D. V. Lightner, R. M. Redman, B. T. Poulos, and B. M. White. 1999b. Taura syndrome virus (TSV) lesion development and the disease cycle in the Pacific white shrimp Penaeus vannamei. Diseases Aquatic Organisms 36: JimCnez, F Acciones para prevenir enfermedades. Pages 1-3 in Proceedings of Camaronicultura For0 International. Mazatliin, 1-3 August Sinaloa, Mtxico. Jimenez, R Sindrome de Taura (resumen) Acuacultura del Ecuador. Revista Especializada de la CBmara Nacional de Acuacultura, Guayaquil, Ecuador. December 1:l-16. Lightner, D. V Epizootiology, distribution and the impact on international trade of two penaeid shrimp viruses in the Americas. Revue Scientifique et Office International des Epizooties 15: Lightner, D. V The penaeid shrimp virus TSV, IHHNV, WSSV and YHV Current status in the Americas, available diagnosis methods and management strategies. Journal of Applied Aquaculture 9: Lightner, D. V., R. M. Redman, K. W. Hasson, and C. R. Pantoja Taura syndrome in Penaeus vannamei (Crustacea: Decapoda): gross signs, histopathology and ultrastructure. Diseases Aquatic Organisms Overstreet, R. M., D. V. Lightner, K. W. Hasson, S. McIIwain, and J. M. Lotz Susceptibility to Taura Syndrome Virus of some Penaeid Shrimp Species Native to the Gulf of Mtxico and the southeastern United States. Journal of Invertebrate Pathology 69: PCrez Farfante, I. and B.F. Kensley Penaeoid and sergostoid shrimps and prawns of the world: keys and diagnoses for the families and genera. Mtmorial Mustum National d Histoire Naturelle 175: Zarain, H. M. and V. F. Ascencio Taura syndrome in Mtxico: follow-up study in shrimp farms of Sinaloa. Aquaculture 193: 1-9.