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Current Trends in Biotechnology and PharmacyVol. 5 (1) 1011-1020 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)10153. Negative Staining : The result of negativestaining of the pellet from the filtrate of diseasedPenaeus monodon epidermis is shown in fig. 4.Viral particles with rod shaped morphology canbe seen.margination and chromatin condensation were alsoevident at lower level (Fig. 5d).Fig. 4 shows rod shaped viral particles by negativestaining in epidermal homogenate.4. Histopathology : Histo-pathological changesin foregut, gills, hepatopancreas and muscle fromtiger shrimp naturally infected with white spotsyndrome were observed. The most pronouncedchanges were observed in gut epithelia. The cellsexhibited hypertrophied nuclei with eosinophilicto basophilic inclusion bodies. Large number ofnuclei demonstrated central chromatin diminutionand margination. Complete condensation of nucleiwas also observed (Fig. 5b). Pyknosis andkaryorrhexis were evident with the necrosis oftissue leading to changes in the normalarchitecture of the tissues. The WSSV infectionin gills was indicated by hypertrophied nuclei,basophilic inclusion bodies and completecondensation of nuclei. Chromatin marginationwas detected at lower level (Fig. 5a). Pyknosisand karyorrhexis were more pronounced inconnective tissue compartment ofhepatopancreas; though damage to the tubularepithelial cells of hepatopancreas was not clearlyevident (Fig. 5c). The muscle tissue showedsevere necrosis, fragmentation and haemocyticinfiltration. Nuclear hypertrophy, chromatinFig. 5a to 5d tissues of P. monodon infected with WSSV.The histopathological changes like hypertrophied nucleus(HY), pyknosis (P), chromatin margination (CM),karyorrhexis (K) and necrosis (N)-in gills (a); gut (b);hepetopancreas (c); and muscle (d) (Fig. 5a to d scalebare=50µm).5. SDS PAGE : A clear change in expression ofproteins in muscles and hepatopancreas has beendetected in infected shrimps as compared touninfected ones during SDS-PAGE analysis. Ininfected muscle tissue, four newly expressedproteins were detected (Fig. 6b). Two newlyexpressed proteins are of higher molecularFig. 6a In infected hepatopancreas, lane 1, 2, 3 showsexpression of three proteins 17kDa, 20kDa and 46kDa;lane 5, 6, 7 shows expression of protein from controlsamples and lane 4 is molecular weight marker band.Figure 6b In muscle, Lane 3, 4, 5, 6, 7 shows fournewly express proteins 79kDa, 58kDa, 36kDa and17kDa; Lane 1 is molecular weight marker band andlane 2 is for control sample.Protein expression in WSSV
Current Trends in Biotechnology and PharmacyVol. 5 (1) 1011-1020 January 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1016weight, 79kDa and 58kDa; whereas two newlyexpressed proteins are of low molecular weight,36kDa and 17kDa. Hepatopancreas of infectedshrimps exhibited two new protein bands with lowmolecular weight, 17kDa and 20kDa, as well asa protein band with high molecular weight, 46kDa(Fig. 6a). In both the infected tissues, a newlyexpressed protein with molecular weight 17kDahas been detected. In hepatopancreas, a 62kDaprotein band has not been detected which hasbeen expressed in uninfected shrimps.DiscussionThe outbreak of white spot syndrome incultured penaeid shrimps have been observed inIndia since 1983 (5,17). In Gujarat (India), shrimpculture is being practiced in the coastal farms inthe southern region; but the outbreak of thediseases in these cultured ponds appears not tohave been analyzed systematically. During themonitoring of hygienic condition of theseaquaculture units, P. monodon with themorphological symptoms of WSSV have beendetected. Monitoring of WSSV in the growoutponds has been suggested as one of the healthmonitoring methods in the farming of shrimps (18).Infected shrimps exhibited lethargy, anoxia,opaque musculature, red body colouration andwhite spots on carapace as well as appendages.White spot syndrome virus is the extremelyvirulent, contagious causative agent of white spotsyndrome of shrimp; and it causes high mortalityand affects commercially cultured marine shrimps(2).Under the SEM, the white spots wereobserved as spheres on both inside and outsidesurfaces of the carapace cuticle. White spots ofinside surface were quite larger (25µm to 135µm)as compared to outside surface, where very smallwhite spots (25 to 32µm) have been detected (Fig.2a-2b). The formation of white spot is acharacteristic clinical sign of WSSV infection (1).The crustacean cuticle is secreted by underlyingepidermal cells and mainly made up of chitin,calcium carbonate as well as protein (19).Cuticular epithelial cells are one of the mostpreferred sites of the WSSV and are amongstthe first sites to be infected (10). White spots arereported to derive from the abnormalities ofcuticular epidermis. Blockage of secretaryproducts from cuticular epithelial cells possiblyby WSSV viral infection might have affectedcuticle with coloured spots. Presently observedmorphological analysis of white spots, analyzedby SEM is found to be similar to the morphologicalanalysis of white spots during WSSV infectionreported by Wang et al. (1).The chemical analysis of white spot byEDX analysis with SEM indicates changes in thelevels of certain elements like C, O, P and Ca ascompared to healthy shrimps. Though Wang etal. (1) has reported that the chemical compositionof the white spot is similar to that of normalcuticle; in the present investigation, a clear increasein Ca and O as well as decrease in C in whitespot region has been detected in comparison withhealthy shrimps. Damage to the cuticularepithelium by WSSV infection seems to beresponsible for the change in the chemicalcomposition of white spots.Evidence of histopathologicalmanifestation in the target tissues is one of thecriteria used in the diagnosis of WSSV infection(9). This paper describes the histopathologicalchanges in foregut, gills, muscles andhepatopancreas of WSSV infected P. monodon.The WSSV target tissues are reported to begenerally of ectodermal and mesodermal in originincluding connective and epithelial tissues,hematopoietic nodules, haemocytes, gills,epidermis, foregut, striated muscles and nerves(2,20). In the present investigation, the cells ofinfected gut and gills exhibited nuclearhypertrophy, chromatin diminution andmargination as well as dense basophilicBhatt et al
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