The status of white spot syndrome virus (WSSV) in Islamic Republic of Iran

Item Type article

Authors Afsharnasab, M.; Kakoolaki, S.; Afzali, F.

Download date 01/10/2021 17:29:56

Link to Item http://hdl.handle.net/1834/37498 Iranian Journal of Fisheries Sciences 13(4)1021-1055 2014

The Status of white spot syndrome virus (WSSV) in Islamic Republic of Iran

Afsharnasab, M.1*; Kakoolaki, S.1; Afzali, F.2

Received: September 2013 Accepted: January 2014

Abstract White spot syndrome virus (WSSV), the causative virus of disease, is found in most shrimp farming areas in Iran, and it cause large economic losses to the shrimp farming industry. Shrimp aquaculture is an important industry in Iran and plays an important role in aquaculture production. The shrimp production in 2012 was more than 12000 metric tonnes and it is estimated it will reach 20000 tonnes in 2013.White spot syndrome virus is highly virulent in shrimp farms and can spread quickly and - cause up to 100% mortality within 3-7 days. The virus is a very large, enveloped, double stranded DNA (ds DNA) and assigned by ICTV to a new genus Whispovirus and belong to Nimaviridae family. In Iran WSSV first appeared in Khuzestan Province in the south of Iran and later on it appeared in other provinces such as Bushehr, and Sistan and Baluchestan. The aim of this review is to give current information of WSSV in Iran, host ranges, carriers, biology, clinical signs, histopathology, PCR, with emphasis on the effects of WSSV in shrimp aquaculture.

Keyword: WSSV, Shrimp, Disease, Aquaculture, Iran.

1-Iran Fisheries Research Organization, Aquatic Animal Health Department, P.O. Box: 14965/149. 2-Department of Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia. *Corresponding author’s email: [email protected]. 1022 Afsharnasab et al. The Status of white spot syndrome virus (WSSV) in..

Introduction number of large farms started to come into Iran, with an area of about 1.62 million km2 production. Initial trials were carried out and a population of about 75 million, is with FAO/UNDP assistance in the years located in arid and semiarid region of West 1991-92, producing post-larvae of local Asia. Iran’s aquatic resources include 2,870 species such as Penaeus semisulcatus, P. km of coastline along the Persian Gulf and merguiensis and Metapenaeus affinis and Oman Sea (1880 km), Caspian Sea (990 reviewing favorable areas for shrimp km), (Fig. 1). There are also large culture. The production in 1993 was 16.5 expansions of flat, barren and saline lands tonnes. When the postlarvae of P. monodon stretching along southern coasts of Iran, were imported from Malaysia the which are unsuitable for agricultural production increased steadily until 2012 purposes. About 5,000 ha of such coastal and reached 12000 tonnes (Fig. 2). Iran has land were developed for shrimp culture. ambitious plans for expansion of shrimp The total area allocated for shrimp culture culture industry from 10,000 tonnes in 2002 is about 200,000 ha (Fig. 1) (Tokhmafshan, to 100,000 tonnes by 2020 (Salehi, 2010; 2001). Farmed shrimp production in Iran Afsharnasab, 2012). grew slowly from 1991 until now and a

Figure 1: Map of Iran with area for shrimp culture in the coastal area of the Persian Gulf and Sea of Oman. Iranian Journal of Fisheries Sciences 13(4) 2014 1023

Shrimp production in Iran 25000

20000

15000

10000 Production/ton 5000

0

2002 1993 1996 1997 1998 1999 2000 2001 2001 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 Year

Figure 2: Shrimp production In I. R. Iran.

The expansion of culture of penaeid and routes of entry are very important shrimps in Iran has been accompanied by (Lightner, 1996; Lightner, 2004; recognition of penaeid diseases that are of Afsharnasab, 2007b; Afsharnasab et al., viral etiology. Viral diseases are very 2007b; ). important because some of them are accompanied by serious crop and economic Biology of Agent losses. White spot syndrome virus is an The causative agent of white spot disease is important invasive pathogen for shrimp white spot syndrome virus (WSSV) which (Afsharnasab et al., 2009a; Kakoolaki et is an envelope, double stranded DNA virus. al., 2010; Lightner, 2011).White spot The virion were elliptical to short rods with syndrome virus was reported first from trilamilar envelop that measured as shrimp farms in Iran in 2001 in Khuzestan 248±87×107±11nm and nucleocapsids Province, followed by other shrimp farming were 162±15×59±17nm. It seems that the area in Bushehr Province in 2003 and virus at the end have a tail and it is very 2005, Sistan and Baluchestan, 2005, 2007, important for shrimp reproduction (Fig. 3), 2008 and 2011 in south of Iran (Afsharnasab et al., 2005, OIE, 2006). (Afsharnasab et al., 2007b; Afsharnasab et According to the International Committee al., 2009a; Kakoolaki et al., 2010; on Taxonomy of Viruses (ICTV) and Atlas Kakoolaki et al., 2011b). The WSSV can be of invertebrate virus, the morphology of prevented and its impacts can be mitigated virus is similar to that of SEMBV of WSSD through implementing scientific health group serotype (Flegel, 2006; Afsharnasab management strategies, and application of et al., 2009a; Lightner, 2011). In the 8th principles of biosecurity at the pond, farm, report of the ICTV in 2004, WSSV is national and regional levels. Knowledge assigned as the only member of the genus about nature of the pathogen, its carriers Whispovirus within the Nimaviridae family 1024 Afsharnasab et al. The Status of white spot syndrome virus (WSSV) in..

(OIE, 2006). More than 50 structural and et al., 2002; Zhang et al., 2002; Tsai et al., non structural proteins are found in WSSV 2004; Zhu et al., 2005; Tsai et al., 2006; Xie that most of them are located in the and Yang, 2006; Xie et al., 2006; Zhu et al., envelope and a few are located in the 2006 ), in the nucleocapsid are VP15, capsule. Proteins located in the envelop are: VP35, VP51C, VP60B, VP388, and VP664 VP12, VP19, VP22, VP24, VP28, VP31, (Van Hulten et al., 2002; Tsai et al., 2004; VP36B, VP38A, VP39, VP41, VP41A, Witteveldt et al., 2005; Tsai et al., 2006), VP41B, VP51B, VP52A, VP52B, VP53, and in the capsule are consisted of VP36A, VP53A, VP68, VP110, VP124, VP150, VP39A, and VP95 (Tsai et al., 2006). This VP187, VP281, VP292, and VP466 (Van envelope protein is suggested to bind to the Hulten et al., 2000; Van Hulten et al., shrimp cells, playing a crucial role in the 2001a; Van Hulten et al., 2001b; Huang et viral penetration (Tsai et al., 2006). al., 2002a; Huang et al., 2002b; Van Hulten

Figure 3: Simplified model of WSSV, Courtesy of (Meezanur Rahman, 2007).

Distribution, History, Host and carrier farmed M. japonicus in 1993 (Inouye et al., Since 1992, white spot syndrome disease 1994, Nakano et al., 1994) and the which was reported from China has causative agent was named penaeid rod- overshadowed all other viral diseases. The shaped DNA virus (PRDV) or rod-shaped white spot disease (WSD) is also reported nuclear virus of M. japonicus (RV-PJ). from all countries in Asia and America. Later, outbreaks of viral disease with White spot disease caused by WSSV similar gross signs caused by similar rod- emerged in east Asia in 1992–1993 and it shaped viruses were reported from was quickly dispersed with infected seeds elsewhere in Asia and other names were and broodstock across Asian continent to applied: hypodermal and hematopoietic SE Asia and India where it caused a major necrosis baculovirus (HHNBV) in the pandemic, and continues to cause People's Republic of China (Huang et al., significant losses in some regions (Flegel, 1995), white spot baculovirus (WSBV) and 2006; Lightner, 2011). In Japan white spot PmNOBIII in Taipei China (Chou et al., syndrome virus was first reported from 1995), and systemic ectodermal and Iranian Journal of Fisheries Sciences 13(4) 2014 1025 mesodermal baculovirus (SEMBV) or Vaseeharan et al., 2003; Gholamhoseini et PmNOBII in Thailand (Wongteerasupaya al., 2013b). The infection can be et al., 1995). First in July 2001, high transmitted vertically transvarian and mortality occurred in cultured P. indicus horizontally by consumption of infected shrimps in Khuzestan Province, south of tissue (e.g. cannibalism, predation, etc.), Iran by WSD (Tokhmafshan, 2004; and by water born methods. Birds such as Afsharnasab et al., 2009a). High mortality gulls may mechanically transmit infection was reported from all farms in Bushehr between ponds by releasing captured, Province as the main area of shrimp culture moribund or dead shrimp. Also the role of in Iran during 2003, 2004 and 2008. In late farm equipments and people should not be 2007, 2009 and 2011, Guader area in Sistan neglected in mechanical transmission of and Baluchestan faced WSD and high virus particles in heavy presence of virus in mortality occurred in shrimp farms during the environment (Khatibitabar, 2010). these years (Afsharnasab et al., 2008, Afsharnasab et al., 2009a, Salehi, 2010, Techniques for WSSV detection Afsharnasab, 2012). White spot syndrome As mentioned by researchers (Lightner, virus can be detected by PCR methods in 1996; Flegel, 2006; OIE, 2006; carrier and reservoir. As described by Afsharnasab, 2007a; Afsharnasab, 2007b) researchers (Chou et al., 1998, Lightner, different methods such as microscopic 2011, Flegel, 2006) many crabs, lobsters, observation under light, dark field, phase arthropods, shrimps and insects are carriers contrast microscope, bioassay, molecular of viruses. Many of them as well as the methods (PCR, Nested PCR, RT-PCR), shrimps such as P. monodon, P. transmission electron microscopy, semisulcatus, P. merguiensis, Metapenaeus immunological and histopathological ensis, P. indicus, Macrobrachium methods are developed to detect WSSV rosenbergii, P. chinensis, P. penicillatus, infection. The selection of a method is M. japonicus, Litopenaeus vannamei, L. dependent on the purpose. For instance, for stylirostris, P. Aztecus, P. californiensis, L. screening brood stock and nauplii in setiferus, P. duorarum, Trachypenaeus hatcheries, different detection methods are curvirostris, Exopalaemon orientalis, used on WSSV pathogenesis. The Orconectes punctimanus, Procambarus frequently used clinical sign, clarkii , Charybdis feriatus, Portunus histopathological, immunological and pelagicus, Portunus sanguinolentus, Scylla molecular methods are described below. serrata, L. occidentalis, Thalamita sp., Panulirus versicolor, Panulirus Wet mount microscopy penicillatus, Cherax quadricarinatus are The wet mount methods where a fresh very susceptible to virus. In Iran most smear of target tissue, organ or feces is penaeid shrimps such as P. Indicus, P. prepared or stained using 0.05% malachite semisulcatus, P. merguensis are very green for checking the virus (Lightner, susceptible to virus (Mohan et al., 1997; 1996; Flegel, 2006; Afsharnasab, 2007a ,). 1026 Afsharnasab et al. The Status of white spot syndrome virus (WSSV) in..

The impression smear method is used to Afsharnasab, 2007b). Staining of feces and compare diagnostic sensitivity to standard HP smear with 0.05% aqueous malachite histopathological methods applied to the green or rapid Huang’s staining show the diagnosis of WSSV infection in postlarvae inclusion bodies under light microscopy for and Juvenile shrimp (Afsharnasab, 2007a; WSD (Fig. 4) (Huang and Yu, 1995).

Figure4: Wet mount smear (Right) and T-E Huangs (Left) courtesy from Huang and Yu (1995) staining for rapid WSSV inclusion bodies detection (arrow), Mag:1000X.

Clinical sign of WSD rings (Fig. 5c) (Flegel et al., 2004; Flegel, White spot syndrome virus infection in 2006; Afsharnasab, 2007b; Khatibitabar, penaeid shrimps typically cause lethargic 2010; Gholamhoseini et al., 2013a, ,). The behavior in affected animals, cessation of cuticulin is easily separated from feeding followed within a few days by the underlying epidermis, and hepatopancreas appearance of moribund shrimp swimming become yellowish-white with a swollen and near the surface at the edge of ponds (Fig. fragile texture (Figs. 5d, 5e). Cuticular 5a) (Lightner, 1985; Jiravanichpaisal et al., deformities such as broken or withered 2001; Afsharnasab and Akbari, 2005; antennae and damaged rostrum, opaque Flegel, 2006; Afsharnasab, 2007b; abdominal musculature and melanised gills Afsharnasab et al., 2007b, , Kakoolaki et are consistently observed (Fig. 5f). Gills are al., 2011a, ). Pink to reddish-brown infected with epicommensal and fouling discoloration of the body and white spots of organisms, especially with Zoothamnium about 0.5-2mm on the cuticle (Fig. 5b), sp. Thereare 70-100% mortality in white especially on the inner surface of the spot affected farms within 3-7 days after the exoskeleton of cephalothorax and onset of the clinical signs (Fig. 5f) abdomen, are the predominant gross (Lightner, 1996; Chou et al., 1998; lesions observed and spots under light Jiravanichpaisal et al., 2006; Afsharnasab microscopy in fresh squash show melanin et al., 2009a; Lightner et al., 2012 ).

Iranian Journal of Fisheries Sciences 13(4) 2014 1027

Figure 5: Moribund shrimp near the edge of farm and shrimp moribund exhibit the red appearance (Fig. 5a). The comparing infected cuticle with white spot (arrow) and non infected cuticle of shrimp (Fig. 5b). The white spot under light microscopy with melanin area (arrow) and circular rings (Fig. 5c), Mag: 1000X. The comparing infected shrimp with opaque muscles and none infected with transparency muscles (Fig. 5d). The hepatopancreas of infected shrimp with yellow color and fragile texture (arrow) (Fig. 5e). High mortality of shrimp infected with WSSV (Fig. 5f).

Histopathology of WSSV Cowdry type -A inclusion bodies in the Histopathological technique by routine tissues. The tissue section of affected paraffin section stained with H and E is shrimp stained with H and E/Phloxine show adapted in diagnostic procedure (Lightner, intranuclear eosinophilic Cowdry type-A 1996; Afsharnasab, 2007a). This technique inclusion bodies in gills, midgut, cuticular is normally quite labor intensive, due to epidermis, lymphoid organ, hematopoietic detection of the inclusion bodies, which tissue, cecum, hearth and connective tissues may be masked by extraneous material and (Lightner, 1996; Kou et al., 1997; Lo et al., difficult to distinguish. However, this 1997; Afsharnasab et al., 2007a; Lavilla- technique can serve as a criterion to confirm Pitogo et al., 2007; Meng et al., 2009; WSSV. The histopathology of WSSD in Lightner, 2011,) Figs 6a-h). However, the shrimp is dominated by presence of large virus does not infect hepatopancreatic conspicuous intranuclear eosinophilic epithelial cells (HEC), even in moribund 1028 Afsharnasab et al. The Status of white spot syndrome virus (WSSV) in.. specimens (Fig. 6i). Although HEC have no basophilic intranuclear inclusion body. viral inclusion, but the hemocytes are With the progress of infection, the inclusion highly infected, which are infiltrated with body is separated by a halo from marginal hemolymph fluids in interstitial space (Fig. chromatin (Fig. 6k). At advance stage of 6j). The hepatopancreas show cellular infection the nuclei of cell is disintegrated vacuolation resulting in diminution of the and the halo space is seen in cells. In tubule lumen (Fig. 6i). These might be the moribund shrimps, there are also focal to reason why hepatopancreas become multifocal necrosis, evidenced by nuclear swollen and fragile (Afsharnasab et al., hypertrophy and vacuolar space around the 2007b; Afsharnasab et al., 2009a). Early nucleus containing the inclusion (Chou et stage of infection is generalized epithelial al., 1998; Wang et al., 1999; Chakraborty hyperplasia and the hyperplasia cells have et al., 2002; Wang et al., 2002; nuclear hypertrophy with chromatin Balasubramanian et al., 2006; Afsharnasab margination containing eosinophilic to et al., 2007b).

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Figure 6: The gill of P. indicus infected with WSSV and Cowdry type A in the cells (arrows) (Fig. 6a) HandE/Pheloxin, Mag: 1000X. Midgut of shrimp L. vannamei infected with WSSV and abundant Cowdery type A in the cell (arrows) (Fig. 6b) HandE/Pheloxin, Mag: 1000X. The cuticular epithelium of L. vannamei infected with WSSV and advances Cowdery type A (arrows) (Fig. 6c), HandE/Pheloxin, Mag: 1000X. Lymphoid organ of shrimp L. vannamei infected with WSSV and the tissue and hemocyte (Right angle) showed intranuclear Cowdery type A, (Fig. 6d) HandE/Pheloxin, Mag: 1000X. Hematopoeitic tissue of L. vannamei with heavily infected with WSSV and the cells showed intranuclear Cpwdery type A, (Fig. 6e) HandE/Pheloxin, Mag: 1000X. The longitudinal section of cecum with cells infected of WSSV (arrows), (Fig. 6f) HandE/Pheloxin, Mag: 1000X. Section of heart muscles and connective tissue with infected cells (arrows) (Fig. 6gandh),HandE/Pheloxin, Mag: 1000X. Hepatopancratic epithelial cell (HEC) with cellular vacuolization (arrows) and lumen (star) without any infection, (Fig. 6i) HandE/Pheloxin, Mag: 1000X. Hemocyte infected with WSSV in shrimp. L.vannamei SPF, (Fig. 6j) HandE/Pheloxin, Mag: 1000X. Different stage of WSSV in shrimp infected with Cowdry type A inclusion body Courtesy by Flegel (2006) (Fig. 6k), Bar:10µm.

Transmission electron microscopy Under TEM the nuclei of infected cells Transmission electron microscopy (TEM) show slight hypertrophy, chromatin is is a powerful tool in the diagnosis of marginated along the nuclear membrane. WSSV. Ultra thin section of gill, The nucleolus and chromatin are fused, exoskeleton, heart and hepatopancreas causing the central area of nucleus to from shrimp suspected for white spot become thin and hemogenous (Fig. disease are processed for TEM studies. 7)(Wang et al., 1995; Wang et al., 1999; 1030 Afsharnasab et al. The Status of white spot syndrome virus (WSSV) in..

Afsharnasab and Akbari, 2005; Flegel, are revealed ovoid, elongated or elliptical 2006; Lightner et al., 2012; Tang et al., virions, typically non-occluded baculo-like 2013, ). The nuclei are rounded due to gross forms within markedly hypertrophy nuclei hypertrophy and very electron-dense (Fig. 9). The virions, which are both marginated chromatin is embedded in a less longitudinally and crossly cut, are electron-dense zone of granular material measured about 248 ± 87 nm×162 ±15 nm that formed a continuous dark layer (ring- and the nucleocapsids are about 162 ± 15 × zone or RZ) adjacent to the nuclear 59±27 nm, with a well development membrane (Fig. 8). More electron-lucent trilaminar envelope. In some hypertrophied central virogenic stroma (VS) contain nuclei, the immature virus with empty distinct viral envelope material in capsid, nucleocapsid and circular envelope membranous or vesicular form. In some along with fully mature virions are detected cells, the central virogenic stroma is denser, in the nucleoplasm (Fig. 10) (Wang et al., with the appearance of numerous viral 2002; Afsharnasab and Akbari, 2005; particles. The progressive infection cells Flegel, 2006; Lightner et al., 2012).

Figure 7: The cuticlar epithelium cell of shrimp P. indicus infected with WSSV and the chromatine marginated(arrow) and nucles (N) and nuclelus (NU) hyoertrophy, the cytoplasm compressed and has a narrow shape. Lead citrate and uranyl acetate.Scale bar:1.5µm.

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Figure 8: WSSV-infected cuticular epidermal cellscourtesy from Wang etal, (1999). The marginated chromatln is transformed into a dense ring-zone (blank arrows). The central virogenic stroma is less dense, vesicular in form, and shows many viral particles (VP) undergoing assembly. Note a viral nucleosome (arrow) in the nucleus. Scale bar = 1.4 pm. Inset: Higher magnificatonof the viraal particle (*) indicating 2 portions of a capsid separated at the ends (arrows).Lead citrate and uranyl acetate. Scale bar = 340 nm.

Figure 9:Ultrathin section of WSSV showed longitudinal and horizontal view of virus (arrow).Lead citrate and uranyl acetate. Scale bar: 200 nm.

Figure10: High magnification of WSSV virion with showed trilamiar envelop and empty capside (arrow). Lead citrate and uranyl acetate. Scale bar: 230 nm.

1032 Afsharnasab et al. The Status of white spot syndrome virus (WSSV) in..

Polymerase chain reaction deionized water. The thermocycler This method can be a highly sensitive program and electrophoresis are done as means for detection of WSSV in both the mentioned by Saberi et al. (2008). The shrimp tissues and the culture environment. results of PCR to detect the presence of Molecular methods also are used to WSSD genomic DNA is shown in Fig. 11. demonstrate that the organism is definitely A single band of 414 bp fragment is a particular species or strain. In Iran as observed after 30 cycles of PCR mentioned by researchers (Afsharnasab et amplifications of viral genomic of WSSV. al., 2005; Saberi et al., 2008) two pairs of According to the procedure of the kit a primers are designed (CLC Bio software) single band of 414 bp of unknown samples using viral VP24 gen for nested PCR. Also is representative of WSSV in the shrimp a pair of primers is designed for host of examined. The appearance of 809 bp 18SrRNA as a PCR control on both positive shrimp DNA product (false negative and negative samples. Primers: Shri F 5´- control) and 414 bp WSSD DNA product in GTA GGT ACG CCT ACA ATG G-3´, and the positive control confirm the validity of Shri R 5´CCG GAA CTC AAA GAC TTT the results (Afsharnasab et al., 2005; Saberi GGT T-3´ are used as PCR control for et al., 2008) (Fig. 12). sampling of 809 nucleotides from shrimp 18SrRNA GEN. Primers WSS F1 5´-CAC t PCR product (Saberi et al., 2008). DNA CTG GGT TTG ACT ACA ATA-3´, and extraction is done as described by WSS R1 5´-TCT GTT TTT TTC TCT CAT (Lightner, 1996) with some brief GAC-3´ are designed to amplify 785 modifications. 50 mg of mixture of hard nucleotide (product of PCRI) from viral and soft tissues of shrimp are poured into a VP24 gene. Primers: WSS F2 5´- TCC microfuge and 200 µL of lyses buffer AAA CAC AAG TGT GTT GAT C-3´, containing 20 µg Ml-1 proteinase K is added WSS R2 5´AAG ACG CCT ACC CTG to it and Kept in 37ºC overnight. Then, it is TTG AAT C-3´ amplify 414 nucleotides boiled and centrifuged. The supernatant (products of PCRII) of the first PCR containing DNA is transferred to a new tube product (Saberi et al., 2008). DNA and extracted using phenol and chloroform. extraction is done as described by DNA precipitate is dissolved in 50 µL of (Lightner, 1996) with some brief deionized water. The thermocycler modifications. 50 mg of mixture of hard program and electrophoresis are done as and soft tissues of shrimp are poured into a mentioned by Saberi et al. (2008). The microfuge and 200 µL of lyses buffer results of PCR to detect the presence of containing 20 µg Ml-1 proteinase K is added WSSD genomic DNA is shown in Fig. 11. to it and Kept in 37ºC overnight. Then, it is A single band of 414 bp fragment is boiled and centrifuged. The supernatant observed after 30 cycles of PCR containing DNA is transferred to a new tube amplifications of viral genomic of WSSV. and extracted using phenol and chloroform. According to the procedure of the kit a DNA precipitate is dissolved in 50 µL of single band of 414 bp of unknown samples Iranian Journal of Fisheries Sciences 13(4) 2014 1033 is representative of WSSV in the shrimp the positive control confirm the validity of examined. The appearance of 809 bp the results (Saberi et al., 2008, Afsharnasab shrimp DNA product (false negative et al., 2005) (Fig. 12). control) and 414 bp WSSD DNA product in

Figure 11: Electrophoresis pattern of PCR product in positive and negative samples in compare with DNA ladder marker. In negative samples only PCR product of shrimp will observe and in positive samples both of PCR products of virus and shrimp will observe. The shrimp PCR product is a control device for PCR system.

Figure 12: Agarose gel electrophoresis of positive and negative samples. Lane 1 show PCR product of positive sample because PCR product of host and pathogen are seen and lane 3 shows negative sample because only PCR product of host is seen.

1034 Afsharnasab et al. The Status of white spot syndrome virus (WSSV) in..

Other researchers from different areas also MBV (Natividad et al., 2006) or IHHNV introduced different protocol for detection and TSV simultaneously have also been of WSSV. These methods are based on developed (Xie, 2007). A new protocol primers designed against a specific part of called in situ PCR can detect light infection the genome sequence of WSSV. PCR in tissues at early stage of infection (Jian, methods include one step PCR (Lightner, 2005). Another method named loop 1996; Lo et al., 1997), semi nested PCR mediated isothermal amplification (LAMP) (Kiatpathomchai et al., 2001), two step is claimed to have more sensitivity than PCR (Tapay, 1999; Hossain et al., 2004), other PCR protocols. It can detect up to 1 quantitative competitive PCR (Tang, 2000) femtogram (fg) of virus (Kono, 2004). and real time PCR (Durand and Lightner, 2002). One step PCR detects WSSV in Sampling and sample size for study WSSV shrimps containing a substantial Laboratory procedures should comply with concentration of viral DNA which is the Manual of Diagnostic Tests for Aquatic usually the case in animals displaying gross Animals (OIE, 2010). The purpose for signs of disease (Otta, 1999; Jian, 2005). specimen collection is observing the Two step PCR can detect light infections in symptom, study the pathogen, and isolate it brood stock, nauplii, postlarvae and or conduct epidemiological survey. There juveniles (Lo, 1996; Lo, 1997), and recommended minimum number of quantitative PCR can be used for the specimens to collect for diagnosis is 100 for quantification of viral load. The larval stages of most crustaceans; 50 for disadvantages are misdiagnosis (false post larval stages; and 10 for juveniles and positive) (Claydon, 2004; Sritunyalucksana adults, with preference for individuals with et al., 2006), inability to confirm whether signs and/or gross lesions. There are two the detected DNA is infectious or not, the situations in which WSSV infection fact that sensitivity depends on the primer requires detection: For confirmation of used (Hossain et al., 2004), lack of suspect clinical WSD and in targeted localization of the infection in tissues and surveillance (screening) to establish the possible presence of inhibitory factors in infection status of asymptomatic some tissues (false negative) (Shekhar, populations. As mentioned by Lightner 2006). The protocols of multiplex PCR to (1996) the sample size in statistical analysis detect WSSV and other viruses such as should be collected as Table 1. IHHNV (Quéré, 2002), TSV (Tsai, 2002),

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Table 1: Sample size based on assumed pathogen prevalence in a population

Population Size of Sample Needed at Prevalence Size 2% 5% 10% 20% 30% 40% 50%

50 50 35 20 10 7 5 2

100 75 45 23 11 9 7 6 250 110 50 25 10 9 8 7

500 130 55 26 10 9 8 7

1,000 140 55 27 10 9 9 8 1,500 140 55 27 10 9 9 8

2,000 145 60 27 10 9 9 8

4,000 145 60 27 10 9 9 8 10,000 145 60 27 10 9 9 8

>/=100,000 150 60 30 10 9 9 8

Confirmation of suspect clinical WSD spawning, moulting, changes in salinity, For confirmation of a suspected outbreak, temperature or pH, and during plankton animals that are representative of those blooms, (Lightner, 1996; OIE, 2010). showing clinical and/or gross signs should Two-Step PCR is the preferred test and be sampled. Whole animals, haemolymph, follow-up bioassay to confirm the presence gills, stomach, abdominal muscles and of viable virus in PCR-positive samples pleopods provide suitable specimens for may be required (Lightner, 2005; examination. Although dead animals can Sritunyalucksana et al., 2006). Two-step sometimes provide useful diagnostic PCR and sequencing are recommended information, they are often unsuitable for methods as well for declaring freedom of a examination because of the rapid onset of country/zone/compartment, only for postmortem changes (Lightner, 1996; juveniles and adults and possibly PLs. For Afsharnasab, 2007a). There is a higher such purpose, Two-step PCR negative probability of detecting the virus in crabs results are required. Where a two-step PCR than in shrimp. The best life stages of positive result cannot be confirmed by crustaceans for detection are late PL stages, sequencing, this counts also as a negative juveniles and adults. Probability of result (OIE, 2010). detection can be increased by exposure to In non-destructive screening by PCR, it is stressful conditions (e.g. eye-stalk ablation, recommended by OIE aquatic manual 1036 Afsharnasab et al. The Status of white spot syndrome virus (WSSV) in..

(2010) to submit (a small piece of) gill, (a 2000; Chen W, 2012). Shrimp larvae are small aliquot of) haemolymph or (a small produced or captured in water with salinity piece of) pleopod. There is also some of 28 to 35 ppt., but post larval stages are evidence to suggest that an ablated eyestalk often stocked in ponds where salinity is would be a good alternative, provided that much lower. When shrimp postlarvae are the compound eye is removed prior to stocked into ponds, they should be submission since it may contain a PCR acclimated gradually to lower salinity to inhibitor (Vaseeharan et al., 2003). reduce stress and mortality. The Definition of a suspect case of WSD, acclimation rate should not exceed 1 or 2 according to OIE aquatic manual (2010), ppt change in salinity per hour (Boyd, for juvenile and adult shrimps are gross 1990). Two species of shrimp, L. vannamei signs of WSD, which for shrimp at any life (90%) and P. indicus (10%) are commonly stage (larva to adult) is mortality, and for cultured in Iran. Best survival and growth shrimp and crab at any life stage (larva to of L. vannamei is at salinities above 20 ppt, adult) are hypertrophied nuclei in squash but P. indicus will survive and grow well at preparations of gill and/or cuticular higher salinity as mentioned by epithelium; unusual aggregates in (Tokhmafshan, 2001; Afsharnasab et al., hemolymph by dark-field microscopy, 2008). inclusion bodies in histological sections in The smallness of the size of the ponds, target tissues. Suspect cases should first be cause alterations in physico-chemical checked by PCR. If in a previously WSSV- properties, especially salinity and free country/zone/compartment, PCR temperature, which consequently prolong results were positive, then, they should be the culture period in shrimp farms resulting confirmed by sequencing. Histopathology, in few epizootics of WSD in Iran in the last probes and electron microscopy can also be decade (Soltani et al., 1998; Kakoolaki, used to confirm the cases. 2004; Kakoolaki et al., 2011b ). So far, the effective impact of temperature on WSSV Environmental factors and WSSV infection outbreak has not been well known. Rahman Infection and disease caused by WSSV in et al. (2007, 2006) showed that higher both cultured and wild penaeid shrimps are temperature of water with 33°C could be influenced by environment parameters used to control the mortality of WSSV (Afsharnasab et al., 2006; Annies and infected shrimps in the field. Due to the Rosamma, 2007; Afsharnasab et al., effects of temperature on metabolism, 2009b). Consequently, manipulation of growth, survival rate and immunological these parameters can control or eliminate criteria, it becomes one of the most infections, benefiting production of important environmental factors in shrimp cultured penaeids. Salinity, temperature farms (Wyban et al., 1995). Esparza-Leal et and pH are important water quality al. (2010) showed that if days of shrimp variables, but they also strongly affect other culture extend to autumn, the susceptibility water quality variables (Cheng and Chen, to WSSV among exposed shrimps could Iranian Journal of Fisheries Sciences 13(4) 2014 1037 increase. The risk of WSSV outbreak is lower salinities, lesser or greater than the reduced when water temperature goes up normal condition, in which the shrimps are and salinity fluctuation is small (Tendencia exposed to WSV could lead to severe et al., 2010). In contrary to the results of mortality of WSD (Liu et al., 2006; Sahoo et al. (2005), the association between Peinado-Guevara and Lopez-Meyer, 2006). WSSV outbreak and environmental risk The outbreak of the WSD in Iran in 2008 factors such as temperature (Corsin et al., (Sistan and Baluchestan Province) had 2002; Peinado-Guevara and López-Meyer, occurred within a week after a heavy 2006), ammonia (Corsin et al., 2001) and raining which made a rapid change in salinity (Kakoolaki et al., 2011a) are salinity (Afsharnasab et al., 2009b). Water evident . Load of the virus is reduced in L. temperature has a profound effect on vannamei in waters with a temperature of disease expression, with average water 32oC (Granja et al., 2006). As Kakoolaki et temperatures of below ~30°C being al. (2011a) mentioned minimum and conducive to WSD outbreaks. Increasing maximum counts of mortality for shrimps the density of shrimps per pond also can act exposed to WSSV at salinities of 30, 40 as a stressor if the ponds’ environmental and 50 ppt were 3.5, 8.5; 0.5, 4.5 and 1.5, factors are poorly managed. In most cases 7.5, respectively. No mortality was of WSD outbreak in Iran, poor management observed in untreated control groups of 30, in farms is believed to be the main stressor 40 and 50 ppt during the experiment (Fig. for occurrence of the disease. 13). It is concluded that the higher and the

Figure 13: Cumulative mortality of each group after distinct hours post inoculation.

The optimal pH range for most species is that might have acted as stressors as between pH 6.5 and 8.5; outside this range mentioned by Corsin et al., (2001). They direct toxic effects can occur and stress found also that WSD outbreaks were levels are high. High pH and low water preceded or coincided with higher pH and temperature might be the reason for WSSV un-ionized [cb4] ammonia. in shrimps and ultimately mass mortality. Extreme pH conditions of 1.2 and 12.4 In Vietnam, although no significant reduced the infectivity of Heliothis nuclear association was detected with salinity, polyhedrosis virus (Ignoffo and Garcia, alkalinity and other water quality variables 1966), and heat inactivated cytoplasmic- 1038 Afsharnasab et al. The Status of white spot syndrome virus (WSSV) in.. polyhedrosis viruses of Bombyx mori Braak, 2002 #572). In addition crustaceans (Argua et al., 1963) and Colias eurytheme have a humeral defense mechanism that is (Tanada and Chang, 1968). Gudauskas and based on different enzyme production and Canerday (1968) further demonstrated has a main role to defend the animal against reduced infectivity of Heliothis NPV and T. microorganism (Balasubramanian et al., ni NPV when exposed to extreme acid or 2008). The crustacean’s innate immune alkaline conditions, UV light, and heat. In system recognizes molecular patterns contrast, little information is available shared by large groups of pathogens, such about the effects of chemical and physical as beta-glucans from fungi and conditions on the infectivity of aquatic lipopolysaccharides and peptidoglycans shrimp’s Whispovirus. from bacteria. Also reports have shown that beta-glucan, Vitamin C, seaweed extracts Immunostimulants and vaccine (fucoidan) and other immunostimulants Afsharnasab (2008) explained aquatic may improve resistance to WSD crustacean immunity such as shrimp, (Afsharnasab et al., 2010). Several studies marine and fresh crab. He mentioned that have shown that resistance of prawns to the immunosystem of crustaceans is WSSV can be enhanced by exposure to consisted of three defense mechanisms: 1) these compounds. As their efficacy and Physical and chemical defense; 2) Cellular methods of administration become better defense; and 3) Humeral defense. The defined, immunostimulants may be used to physical and chemical defense containing improve the resistance of farmed cuticle and skin, and both have to do with crustaceans to WSSV and other pathogens secretion, but this defense system is not in an attempt to reduce the risk of disease sufficient to prevent enter of particles to the outbreaks. However, any benefit they may body, particularly the crustaceans have confer is likely to be minimal in adverse open circulating system. During ecdysis, environments or in the absence of crustaceans are easy targets for the attack appropriate disease prevention strategies. of organisms, so they need the Although recent studies of crustacean immunosystem to fend. Because immunology suggest some capacity for crustaceans have open circulating system, acquired immunity, but currently no the blood or lymph coagulation is very consistently effective vaccination methods important. The coagulation system helps have been developed. Ghaednia et al. the animal to control the missing lymph (2012) reported the potency of dietary β 1,3 and prevents diseases (Mohajeri et al., and 1,6 glucan (BG), derived from 2011). In crustaceans blood is called Saccharomyces cerevisiae, in stimulating haemolymph and blood cells are called the non-specific immunity of white Indian hemocyte. The hemocyte is comprised of shrimp, P. indicus and improving its hyaline, granular and semigranular cells. resistance to white spot syndrome disease. Each of them has a main role in disease They also reported significant increase of prevention (Kakoolaki et al., 2010{van de parameters such as total hemocyte count Iranian Journal of Fisheries Sciences 13(4) 2014 1039

(THC), differential hemocyte count (DHC), protection of shrimp against the virus. The total plasma protein (TPP), Phagocytic main vaccines used as mentioned in the activity (PA), bacterial clearance efficiency literature consist of VP28 (Van Hulten et (BCE) and bactericidal activity (BE) when al., 2001a; Witteveldt et al., 2004a; P. indicus shrimps (11.32 ±1.20 g) were Witteveldt et al., 2004b),VP19 (Witteveldt immersed in seawater (39 ppt and 25 ±1○C) et al., 2004b), VP15 (Van Hulten et al., containing hot-water extracts of the brown 2001a), DNA vaccines (Rout et al., 2007; alga, Sargassum glaucescens, at 100, 300 Li et al., 2010), or with dsRNA vaccines and 500 mg/l, and Laminaria digitata (Robalino et al., 2006; Kim et al., 2007). In (p<0.05, Ghaednia et al., 2011). Iran, Afsharnasab et al. (2010) explained Dashtyannasab et al. (2009) studied nuclear and non nuclear methods for complementary feedstuff extracts from production of vaccine against WSSV. He Ascophyllum nodosum containing 1% used three methods, Gama radiation, alginic acid as shrimps stimulating immune electron beam and formalin with 50% system for the control of WSSV. The L. protection dose for controlling WSSV in P. vannamei shrimps in larval stages (Z1- indicus shrimps. Their results showed that PL1), post larval stages (PL1-PL10) and Gama radiation has better protection juvenile (from 30th day to 40th day) were against WSSV comparing with other also fed by complimentary feedstuff as methods. control group. Their results showed that the survival rate of exposed groups were Past and Future of WSSV in Iran significantly higher than that of the control According to FAO and GOAL 2001 group (p<0/05). Their results also showed (Valderrama and Anderson, 2011), world that mortality in the exposed groups was production of cultivated shrimp has observed 48 hours later than the control increased steadily since early 1990’s and group. Many articles focused on vaccine reached 4.2 million tonnes in 2013 (Fig. use for controlling WSSV in shrimp. DNA 14). It is also known that the world shrimp vaccination, recombinant and oral vaccine, fishery is not growing, while the demand and gene therapy are some of the methods for shrimp is increasing steadily. Only for WSSV vaccine use in shrimp. Many aquaculture can meet this increasing structural viral envelop proteins from demand. WSSV genome are used as vaccine for 1040 Afsharnasab et al. The Status of white spot syndrome virus (WSSV) in..

Figure 14: GOAL 2011 survey world shrimp aquaculture (Including M. rosenbergii) by specious. Source: FAO, 2011; GOAL, 2011.

Figure 15: Issue and challenge in shrimp aquaculture in 2011 in all countries. Source: FAO, 2011;GOAL, 2011.

According FAO and GOAL (Valderrama loss of approximately 22% in a single year and Anderson, 2011) shrimp production in due to diseases. Given a total production of the world in the future will face many issues 4.2 million tonnes in 2013 with a value of and challenges (Fig. 15) that the main and roughly $ 7 per kg, this is translated into an extremely important issue of them is estimate of about US$ 6 billion loss in a disease. Despite the explosive growth in year. This is probably a conservative world production of cultivated shrimp, estimate, since farms with very bad results there have also been staggering, periodic may not have responded to the survey. losses due to disease. A global shrimp Thus, a conservative estimate for the total survey by the Global Aquaculture Alliance loss to disease over the past 15 years may (GAA) in 2011 revealed a rough overall be in the order of $ 50 billion. This Iranian Journal of Fisheries Sciences 13(4) 2014 1041 illustrates the importance of disease control stocking density, resulting in great to the industry. With respect to disease economic loss. In 2007, an outbreak of agents, the GAA survey revealed that 60% disease of cultured Indian white shrimp of losses were attributed to viruses and the occurred in semi-intensive farms in shrimp rest from bacteria, fungi and protozoa. In sites of Goader (Sistan and Baluchestan Iran the shrimp production and impact of Province), southeastern Iran. During these WSSV in this industry is divided into three years Iran Fisheries Research Organization phases (Fig. 16). At the first phase, from (IFRO) imported Specific Pathogen Free L. 1992 until 2001, shrimp cultivation systems vannamei from Hawaii and used this were semi extensive and stocking densities species instead of P. indicus (Afsharnasab were low, disease problems were relatively et al., 2008). Many research projects have few and production were relatively low. In been done on this species and the results these years, there were few disease showed that this is a good shrimp species specialists available to help shrimp farmers, for culture in Iran’s conditions and diagnostic capabilities in most regions (Afsharnasab et al., 2007c; Afsharnasab et were limited. This was a vulnerable al., 2008). Serious WSD disease outbreaks situation as the industry was growing revealed during 2002 - 2008 that the shrimp exponentially with trends towards industry was forced to be better prepared increasing farm densities in suitable with more knowledge about shrimps and farming areas and increasing rearing their pathogens so that disease prevention (stocking) intensity in individual ponds. In methods could be improved. This need these years shrimp production increased shifted attention to biosecurity, that is, steadily and from 16 tonnes in 1991 the possible methods of cultivating shrimp in total production reached 7600 tonnes in restricted systems designed to prevent entry 2001. The second phase started from 2001 of potential pathogens. The industry also (Fig. 16), explosive and large-scale shrimp realized that there is a good number of production of shrimp was made possible by disease outbreaks originated from careless development of eyestalk ablation technique transboundary movement of contaminated to stimulate maturation of captured female but grossly normal aquaculture stocks. broodstock and stocking density gradually More than any other problem, the WSSV increased, especially in Khuzestan pandemic served as a “wake up” call that Province. As mentioned by Afsharnasab et shocked the industry into concerted actions. al., (2005) and (2012) and Salehi (2010) an The catastrophic losses had serious impacts outbreak of WSD of cultured Indian white on whole national economy as well. shrimp occurred in 2001 in semi-intensive The third phase of shrimp culture in Iran farms in Abadan (Khuzestan Province), started from 2008 until now (Fig. 16), southwestern Iran, where it caused losses of during which the production increased almost 100 percent of the production. Later rapidly and reached to an estimated 20000 WSSV disease occurred in all shrimp sites tonnes of L. vannamei in 2013 in Bushehr Province, where it severely (Afsharnasab, 2012). We believe that the affected small-scale farms practicing high Iranian shrimp industry will be dominated 1042 Afsharnasab et al. The Status of white spot syndrome virus (WSSV) in.. by cultivation of domesticated lines of achieved by following Good Aquaculture shrimp that are free of most, if not all, of the Practices recommended by the Global significant shrimp diseases. Most of the Aquaculture Alliance. Iran is also going to stocks used will also be improved by produce SPF L. vannamei shrimp and the genetic selections for growth rate and other combination of using SPF stocks and desirable traits like disease tolerance. We proper management would greatly reduce already know from experience with L. the risk of disease outbreaks and essentially vannamei that such stocks are highly eliminate the need for chemotherapy in the successful when reared with good future. biosecurity and management of feed and pond environment. The latter can be

Figure 16: Past and future impact of WSSV in shrimp industry of Iran. Discussion white spot disease affected farms within 3- The clinical signs in white spot disease 7 days after onset of the clinical signs, affected shrimps in Iran, closely match which was considered a characteristic of published descriptions of white spot disease WSD (Wang et al., 1995; such as white spots on the carapace and Wongteerasupaya et al., 1995; Wang et al., appendages, and signs of lethargic and 1999). yellowish discoloration of hepatopancreas The diagnosis of WSSV by gross sign is with 70-100% mortality (Chou et al., 1995; difficult, because some diseases show the Wang et al., 1995; Wongteerasupaya et al., same symptoms. The IHHNV is a viral 1995; Wang et al., 1999). Additionally, shrimp disease that show white spots in the cuticular deformities such as broken or body but the white spots in IHHNV first withered antennae and damage rostrum, appear on the body and then on the carapace opaque abdominal musculature and (Afsharnasab, 2007b). High pH and melanised gill were observed in these vibriosis also show white spots on the shrimps. There was 70-100% mortality in cuticle of shrimp (Afsharnasab, 2007b). Iranian Journal of Fisheries Sciences 13(4) 2014 1043

Diseased shrimp show widespread focal to must be disinfected, and then passed to the diffused cellular degenerations and unclear drainage system for storage. (3) If there is hypertrophy in most tissues of ectodermal no drainage system, affected shrimps and and mesodermal origin. Additionally the pond water must be disinfected at once, necrosis is observed in the hepatopancreas. and kept in the pond for a few days (5-10 Necrosis, hemocytic infiltration and days) before discarding. An important hypertrophied nuclei eosinophilic to point: do not directly release diseased basophilic inclusion bodies within the shrimp and pond water into public hypertrophied nuclei of affected cells, are environment as it plays an important role in considered as characteristics of white spot spread of the disease. disease (Chou et al., 1995; Wang et al., Because treatment of viral diseases in grow 1999; Pazir et al., 2011). out ponds is expensive, best is to prevent There is no treatment for viral diseases, the occurrence of these diseases. Preventing and it’s latent in healthy shrimp. The diseases are much more economic then disease agents are virus that are latent and providing expensive treatments following a are also found in the adult stages. If the disease outbreak. However, there is not a shrimp is unhealthy or in a stressed single, ideal and universal preventive condition, caused by overcrowding, program that can be applied in every diseases may occur (Afsharnasab et al., procedure. With respect to prevention we 2009a). As vaccines are not available for recommended: shrimp viruses and use of 1. Development of a rapid, simple and immunostimulants is far fetched in accurate method for detection of virus practical scale at the moment in shrimp infection such as PCR diagnostic kit. At ponds (Afsharnasab et al., 2010), thus present, MBV and WSSV infections may disinfectants are useful to eliminate viruses be detected for their characteristic in culture systems. Based on the present inclusions in either wet mounts or study, proper management of pond water histological preparations of hepatopancreas and infected shrimps are recommended as or midgut contents. follows. (1) Drainage system with long 2. Establish virus-free broodstocks drainage canal, and sedimentation and (SPF) in order to hatch free postlarvae for treatment ponds should be established as a shrimp production farms. part of farm construction. During normal 3. Low stocking density, intensive culture period, all discharged water must culture systems encourage development pass through drainage system because and transmission of many diseases numerous zooplankton including small including viruses. wild shrimp and copepods often coexist 4. Proper feeding management, feeds with cultured shrimp in the ponds, and they must be controlled to a minimum. feed were also found to be carriers of viruses. residues which increase organic wastes (2) Shrimps confirmed with viruses such as which adversely affect the water quality MBV and WSSV infection should be and the production removed using a seine net. The pond water 5. Use of disinfectants or chemicals to 1044 Afsharnasab et al. The Status of white spot syndrome virus (WSSV) in.. prevent or treat bacterial sepsis or protozoal establish practical guidelines and epicommensals if necessary. regulations for farmers and iv) conduct 6. Educate shrimp culturists and farm regular monitoring on the use of chemicals. technicians on disease epizootiology and preventive measures. References We strongly believe that many of the Afsharnasab, M., 2007a. Diagnosis disease problems encountered in methods in shrimp diseases. Tehran: aquaculture can be avoided with good Iran Fisheries Research Organization. management practices. In Iran the two p.175. major causes of problems for shrimp Afsharnasab, M.; 2007b. Viral Diseases farmers are due to poor management of Shrimp, Iran. Iran Fisheries practices and environmental stresses. Research Organization. p.210. Management problems are usually solved Afsharnasab, M., 2008. Immunology of through assistance provided by government crustacean with emphasis in shrimp extension workers or from technical sales and defense mechanism against white staff of various feed companies. spot syndrome virus. Scientific Report. Environmental problems are more difficult Iran Fisheries Research Organization. to solve especially when they result from 86/708.: p.53. natural phenomena such as red tide. When Afsharnasab, M., 2012. Review of WSD environmental problems are caused by poor in Iran (Past, Present, Future) and the farming practices, then it would be effect on shrimp production. Paper necessary to gain co-operation among all present in 17th Iranian Veterinary farmers in the area if disaster is to be Congress,Theran,Iran 28-30 Aprill. avoided. And in some cases, government Afsharnasab, M. and Akbari, S., 2005. legislation may be necessary to limit Identification of white spot syndrome practices that may threaten sustainability of disease (WSSD) by electron the industry. Regarding sustainable microscopy in Penaeus indicus. shrimp aquaculture in Iran, regulations are PAJOUHESH-VA-SAZANDEGI .66, required to control stocking density, use of 8-13. chemicals and drugs in aquaculture, pond Afsharnasab, M., Akbari, S., Shariff, M., design, control water inlets and outlets, and Yousef, F. and Hassan, D., 2006. shrimp feeding regimes. Currently Iranian Effect of different pH and salinity Fisheries Organization (Shilat) with levels on the viabilty of Penaeus assistance of the government is drafting monodon Baculovirus (US (MBV) in legislations for the aquaculture industry. In Penaeus semisulcatus. Iranian Journal the larger interest of developing of a of Fisheries Sciences, 6, 1-18. sustainable shrimp culture industry in Iran, Afsharnasab, M., Dashtiannasab, A. and the following measures are recommended: Yeganeh, V., 2007a. Assessing i) establish diagnostic laboratories, ii) pathogenesis of the white spot provide training for the farmers, iii) syndrome virus (WSSD) in the Iranian Journal of Fisheries Sciences 13(4) 2014 1045

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