Immune responses of Fenneropenaeus chinensis against white spot syndrome virus after oral delivery of VP28 using Bacillus subtilis as vehicles

Fish & Shellfish Immunology 28 (2010) 49–55

Contents lists available at ScienceDirect

Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi

Immune responses of Fenneropenaeus chinensis against white spot syndrome virus after oral delivery of VP28 using Bacillus subtilis as vehicles Ling-Lin Fu a, Jiang-Bing Shuai c, Zi-Rong Xu b, Jian-Rong Li a, *, Wei-Fen Li b, ** a

Food Safety Key Laboratory of Zhejiang Province, School of Food Science and Biotechnology, Zhejiang Gongshang University, 149 Jiao Gong Road, Hangzhou 310035, PR China Key Laboratory of Molecular Animal Nutrition, Ministry of Education, College of Animal Sciences, Zhejiang University, 164 Qiu Tao North Road, Hangzhou 310029, PR China c Zhejiang Entry & Exit Inspection and Quarantine Bureau, Hangzhou 310012, PR China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 July 2009 Received in revised form 4 September 2009 Accepted 20 September 2009 Available online 1 October 2009

The protective efficacy of oral administration of VP28 using Bacillus subtilis as vehicles (rVP28-bs) in shrimp, Fenneropenaeus chinensis, upon challenge with white spot syndrome virus (WSSV) was investigated. The calculated relative percent survival (RPS) value of rVP28-bs fed shrimp was 83.3% when challenged on the 14th day post-administration, which is significantly higher (p < 0.001) than that of the group administered recombinant Escherichia coli over-expressing rVP28 (rVP28-e21). After immunization, activities of phenoloxidase (PO), superoxide dismutase (SOD) and inducible nitric oxide synthase (iNOS) in hemolymph were analyzed. It was found that the supplementation of rVP28-bs into shrimp food pellets resulted in the most pronounced increase of iNOS activity (p < 0.001), but had the least influence on activities of PO and SOD. Besides, in the shrimp orally administered with rVP28-bs, the caspase-3 activity was one-fifth that of the control, though the signs of apoptosis (chromatin margination, nuclear fragmentation and apoptotic bodies) could not be observed by transmission electron microscope (TEM). These results suggest that by oral delivery of rVP28-bs, shrimp showed significant resistance to WSSV and an effect on the innate immune system of shrimp. The remarkably enhanced level of iNOS after rVP28-bs administration might be responsible for antiviral defense in shrimp. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: White spot syndrome virus Fenneropenaeus chinensis Bacillus subtilis VP28 Oral delivery Protection Immune response

1. Introduction White spot syndrome virus (WSSV), which was first discovered in northern Taiwan around 1992, is currently the most serious viral pathogen of shrimp worldwide [1]. It is an ellipsoid to bacilliform, enveloped particle with a tail-like appendage at one end, which belongs to a new virus family, the Nimaviridae [2]. Although considerable progress has been made in WSSV molecular characterization [3–5] as well as potential anti-WSSV strategies based on shrimp defense system [6,7], no adequate treatment is available. It was mostly attributed to two obstacles: the poor understanding of WSSV infection and replication mechanisms as well as the molecular process of the shrimp immune responses against this virus. Generally, shrimp are believed to have an innate immune system efficient to protect and preserve themselves from intruding pathogens [8]. The innate defense system of shrimp comprises cellular and humoral responses, mainly including the prophenoloxidase (proPO) system, phagocytosis, apoptosis as well as generation of microbicidal molecules such as antiviral peptides, * Corresponding author. Tel./fax: þ86 571 88056656. ** Corresponding author. Tel.: þ86 571 86986730; fax: þ86 571 86994963. E-mail addresses: [email protected] (J.-R. Li), wfl[email protected] (W.-F. Li). 1050-4648/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2009.09.016

reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (RNIs) [9–12]. Crustaceans are considered to lack a true adaptive immune system, but now it is reported that memory responses could be inducible using either inactivated pathogens or recombinant proteins against WSSV [6,7,13,14]. Thus, the development of subunit ‘‘vaccines’’ based on WSSV structural proteins would be desirable and feasible as these proteins are the first molecules to interact with the host, and they therefore play critical roles in the triggering of host defenses. VP28, one of the major envelope proteins of WSSV, has been shown to be a potential WSSV ‘‘vaccine’’ candidate [6,15–19]. However, most of the reported WSSV ‘‘vaccine’’ agents were in injectable forms, with potential risky and costly/time-consuming administration which made it practically unfeasible in shrimp farming. In recent years, Bacillus subtilis, the well-known host for industrial enzyme production, has been explored as a tool for expression and delivery of antigen proteins, due to the immense capacity of secreting proteins, lack of pathogenicity and function of immune stimulation [14,20]. In a previous paper, we reported that the recombinant B. subtilis strain with the ability of high-level secretion of rVP28 can evoke protection of crayfish against WSSV [14]. The purpose of this study was to evaluate the resistance against WSSV of shrimp, Fenneropenaeus chinensis, by oral delivery

50

L.-L. Fu et al. / Fish & Shellfish Immunology 28 (2010) 49–55

of VP28 using B. subtilis as vehicles. Moreover, the humoral and cellular responses after immunization were also investigated.

tested for the presence of WSSV by PCR [23]. Mortality was recorded for 25 days post-challenge.

2. Materials and methods

2.5. Determination of immunological parameters

2.1. Recombinant strains

Phenoloxidase (PO) activity was measured spectrophotometrically at 490 nm by recording the formation of dopachrome produced from L-3,4-dihydroxyphenylalanine (L-DOPA) according to the reported method [24]. Briefly, a sample of 100 ml treated hemolymph was incubated with 50 ml trypsin (Sigma, 1 mg ml-1) at 25  C for 10 min. Then, 50 ml DOPA was added followed by 800 ml of cacodylate buffer 5 min later. The optical density at 490 nm was measured using a UV-2550 spectrophotometer (SHIMADZU). SOD activity was determined by the ability to inhibit superoxide radical-dependent reactions using a Superoxide Dismutase Activity Assay Kit (BioVision, USA). Briefly, the 10 ml aliquot was placed in a microplate (96-wells) that contained 200 ml of 2-(4-iodophenyl)3-(4-nitrophenyl)-5-(2,4-disulfophenyl)- 2H-tetrazolium (WST) Solution. After addition of 20 ml Enzyme (xanthine oxidase) Solution, the mixture was incubated at 37  C for 20 min. The optical density was measured at 450 nm using a microplate reader. One unit of SOD was defined as the amount required to inhibit the rate of xanthine reduction by 50%. The total NOS activity in hemocytes of immunized and control shrimp was analyzed using L-citrulline based on the principle that NOS can catalyze L-arginine to generate L-citrulline and NO quantitatively by the method of Jiang et al. [12]. After supplementation of the inducible NOS (iNOS) inhibitor 1400 W (Sigma), the identical procedure was used to calculate the iNOS activity of shrimp hemocytes.

As we reported, the vp28 gene was introduced into B. subtilis WB600 (DnprE DaprE Depr Dbpf Dmpr DnprB) to form the recombinant strain rVP28-bs which can secrete rVP28 at a highlevel [14]. The rVP28-bs was incubated in an optimized culture medium (10 g l1 Glucose, 15 g l1 peptone, 1.5 g l1 beef extract, 1.5 g l1 yeast extract, 5 g l1 NaCl, 3.5 g l1 KH2PO4 and 2.5 g l1 CaCO3) at 35  C. Sporulation of B. subtilis was induced in DSM (Difco-sporulation media) using the exhaustion method as described [21]. The spore suspension was titrated immediately for CFU ml1 before freezing at 20  C. The recombinant Escherichia coli BL 21 harboring rVP28 (rVP28-e21) was constructed and stored in our laboratory for a comparative assay. 2.2. Coating of feed pellets The rVP28-bs suspension containing 1010 spores was coated with 0.01 g of commercial pellets (Crown Feed Co. Ltd., China). The bacteria were mixed with the food pellets, and incubated on ice for 15 min to allow absorption of the bacterial suspension, and coated with cod liver oil to prevent dispersion of the spores in the water. Coating with phosphate-buffered saline (pBS) was performed for the positive and negative controls. The rVP28-e21 and B. subtilis WB600 strains were also coated for the comparative groups. The prepared food pellets were stored at 4  C until further use.

2.6. Caspase-3 activity assay 2.3. Shrimp culture Live Chinese shrimp (F. chinensis), approximately 25 g in body weight, were purchased from a local shrimp farm and tested for the presence of WSSV by PCR to ensure that they were WSSV-free. Shrimp of each group were stocked in 100-l aquaria of sandfiltered, ozone-treated and flow-through salt water (30&) heated to 20  1  C. They were acclimated to the laboratory conditions for 7 days and fed with commercial or treated feed at 5% of body weight per day in the whole experimental period. 2.4. Oral administration of recombinant strains harboring rVP28 After acclimatization for a week, shrimp were divided into four sets, and each set with three groups. There were 25 shrimp in triplicate in each group. The four sets were treated by feeding rVP28-e21-, rVP28-bs-, B. subtilis- and pBS buffer-coated food pellets, respectively, for 20 days. On the 3rd day after 20-day feeding, hemolymph was collected from 5 shrimp of each group for analysis of immunological parameters and the remaining 20 shrimp were challenged with WSSV or pBS buffer. The same procedure was performed on the 14th and 28th day after the 20 day oral administration. The scheme of sampling and challenge is shown in Table 1. Virus stocks were purified from gill tissues by 30% sucrose differential centrifugation [22] and stored at 80  C. Ahead of the oral administration experiment, in vivo titration of WSSV was conducted as previously described [14]. Shrimp were challenged with different virus dilutions and the obtained time-mortality relationship was used to determine the desired challenge pressure of c. 75% mortality for the subsequent experiments. In order to mimic the natural route of infection and the initial situation in a pond, shrimp were challenged via immersion. Dead shrimp were

Two groups of shrimp were fed with rVP28-bs- and pBS coated food pellets for 20 days and then challenged with WSSV. In each group, fourteen shrimp in triplicate were included. At the 2nd, 3rd and 4th day post-challenge, hemolymph from each group was collected for detection of caspase-3 activity. The shrimp taken from the WSSV-challenged stock looked normal on the 2nd day postchallenge, however, the individuals of WSSV challenge were moribund in the pBS group after 3 and 4 days post-challenge which exhibited lethargy, red body coloration, as well as a few white spots on the carapace. The caspase-3 activity was detected by a Caspase 3 Activity Assay Kit (Millipore) according to the manufacturer’s instructions. One unit is the amount of enzyme that will cleave 1.0 nmol of the colorimetric substrate Ac-DEVD-pNA per hour at 37  C under saturated substrate concentrations. 2.7. Histological examination At the 2nd day post-challenge, six randomly selected shrimp from rVP28-bs and 6 individuals from pBS group were sacrificed by stunning in ice water and fixed with Davidson’s fixative for light microscopy (LM) and 2.5% glutaraldehyde fixed samples were stained with uranyl acetate and lead citrate for examination with a JEM-1200EX transmission electron microscope (TEM). 2.8. Statistical analysis Statistical calculations were performed using SPSS (version 9) software. Significant difference was indicated by one-way ANOVA (p < 0.05) analysis. The protection against WSSV after administration was calculated as the relative percent survival (RPS) [(1oral administrated group mortality /control group mortality)100] [25].

L.-L. Fu et al. / Fish & Shellfish Immunology 28 (2010) 49–55

51

Table 1 The scheme of sampling and challenge after 20-day feeding. Sampling & Challenge time points

Group

rVP28-e21

No. of Shrimp

25(3)

On the 3rd day post-administration

Sampling WSSV Challenge pBS Challenge

5(3) 20(3) –

On the 14th day post-administration

Sampling WSSV Challenge pBS Challenge

On the 28th day post-administration

Sampling WSSV Challenge pBS Challenge

rVP28-bs

25(3)

25(3)

25(3)

Bs 25(3)

25(3)

5(3) 20(3) – 5(3) 20(3) –

pBS

25(3)

25(3)

5(3) 20(3) – 5(3) 20(3) –

5(3) 20(3) –

25(3)

25(3)

25(3)

5(3) 20(2) 20 5(3) 20(3) –

5(3) 20(3) –

3. Results

25(3)

5(3) 20(2) 20 5(3) 20(3) –

5(3) 20(2) 20

than the theoretical size of 22 kDa (Fig. 1A,B). The rVP28 protein could be directly secreted into the culture medium from the rVP28bs strain (Fig. 1A), while expression was confirmed to occur inside the cells of the rVP28-e21 strain (Fig. 1B). For optimization of the rVP28 secretion level in B. subtilis WB600, a culture medium was selected instead of LB medium (data not shown). On extrapolation by BandScan (Glyko) from the SDS-PAGE,

3.1. Analysis of expressed rVP28 in recombinant strains The vp28 was expressed in both recombinant strains, rVP28-bs and rVP28-e21, evident as a distinct band at a position equivalent to a molecular mass of w28 kDa by SDS-PAGE and significantly higher

A

B

Amount of secreted rVP28 (mg/l)

C

48

optimized culture medium LB medium

40 32 24 16 8 0 0

3

6

9

12

15

18

21

24

27

Time (h) LB medium 4

8

10

12

15

18

21

22

23

24

26

(h)

Optimized culture medium

Fig. 1. Analysis of expressed rVP28 in B. subtilis WB600 and E. coli BL21. (A) Lane 1: Supernatant culture of B. subtilis WB600 harboring empty vector pBS-H1 at 21 h (negative control); Lane 2: Coomassie brilliant blue-stained Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of supernatant of rVP28-bs strain at 21 h. Arrow indicates the secreted rVP28 protein. (B) Lane 1: Expression of rVP28 in E. coli BL21. Lane 2: E. coli BL21 harboring pET-30a vector (negative control). Arrow indicates the over-expressed rVP28. (C) The approximate amount of secreted VP28 in different culture media by time-course analysis using BandScan software.

52

L.-L. Fu et al. / Fish & Shellfish Immunology 28 (2010) 49–55

the amount of rVP28 in the optimized culture medium accumulated to a maximum level (46.3 mg l1) after 21 h of cultivation (Fig. 1C), which was an increase of 30.1% compared with that in LB medium (35.6 mg l1). 3.2. Increased protection against WSSV by rVP28-bs delivery Oral administration of rVP28-bs and rVP28-e21 strains, both harboring the WSSV envelope protein VP28, resulted in a significantly lower cumulative mortality at different challenge time

cumulative mortality (%)

A

points compared with the positive control groups (Fig. 2). Surprisingly, the cumulative mortalities of rVP28-bs group were 23.3  3.3%, 16.7  4.4% and 28.3  1.7% at the challenge points of 3, 14 and 28 days post-administration, respectively (Fig. 2A–C), which were all lower than that of the rVP28-e21 group. The RPS value of rVP28-bs group (83.3%) reached the highest (p < 0.001) when shrimp were challenged at the 14th day postadministration, compared with the positive control (Fig. 2B). Furthermore, by oral delivery of B. subtilis in the form of spores, the number of survivors was also increased after WSSV challenge

100 90 80

rVP28-e21 rVP28-bs Bs positive control negative control

70 60 50 40 30

**

20 10 0 0

3

6

9

12

15

18

21

24

27

Days post challenge

cumulative mortality (%)

B

100

rVP28-e21 rVP28-bs Bs positive control negative control

90 80 70 60 50 40 30 20

**

10 0 0

3

6

9

12

15

18

21

24

27

Days post challenge

cumulative mortality (%)

C 100 rVP28-e21 rVP28-bs Bs positive control negative control

90 80 70 60 50 40 30

*

20 10 0 0

3

6

9

12

15

18

21

24

27

Days post challenge Fig. 2. Protective effects against WSSV of recombinant VP28 by oral delivery of spores of rVP28-bs strain to F. chinensis. Cumulative mortality rates of shrimp from the experimental groups were indicated. Shrimp were challenged 3 days (A), 14 days (C) and 28 days (C) after cessation of feeding coated pellets. The error bars indicate standard error of the mean (SEM). Statistical significance was marked by the star.

L.-L. Fu et al. / Fish & Shellfish Immunology 28 (2010) 49–55

leading to RPS values of 19.7%, 20.0% and 12.3% challenged at 3, 14 and 28 days post-administration, respectively, compared with positive controls but these were not significantly different (Fig. 2). The negative control groups showed no mortality. Three randomly selected survivors from each group were tested for WSSV by PCR [23] and tested negative. 3.3. Detection of immune-related enzyme activities The PO, SOD and iNOS activities of F. chinensis fed rVP28-bs were found to be higher than all the other groups at the different time

A

2.4

rVP28-e21

rVP28-bs

Bs

Phenoloxidase activity (O.D. 490 nm)

3.4. Suppression of WSSV-induced apoptosis by rVP28-bs

2.0 1.6 1.2 0.8 0.4

3

SOD activity (U ml-1)

points (3rd, 14th and 28th day post-administration), which reached the highest level after 14 days post-administration (Fig. 3). The values of PO and SOD in rVP28-bs group were slightly higher than those in Bs group, but no significant differences were observed (p > 0.05) (Fig. 3A,B). However, there was a significant difference in the iNOS value (p < 0.001) between rVP28-bs and Bs groups (Fig. 3C). The activities of PO and SOD in the rVP28-e21 treated shrimp were lower than that in the Bs group at all the time points (not significantly different) (Fig. 3A,B), while a significant difference in the iNOS value (p < 0.001) was noted in the above two groups (Fig. 3C). On the whole, all the values of immunological parameters of rVP28-bs group were demonstrated to be significantly (p < 0.05) higher than those of rVP28-e21 group (Fig. 3).

pBS

0.0

B

53

1.0

14 28 Days post administration

rVP28-e21

rVP28-bs

Bs

pBS

0.8

The caspase-3 activity of F. chinensis fed with pBS coated pellets was about 5-fold higher than that of rVP28-bs group on day 3 postWSSV challenge (Fig. 4). Light microscope examination of surviving shrimp in pBS (control) group on day 2 post-challenge confirmed signs of apoptosis (i.e. chromatin margination, nuclear fragmentation in hepatopancreas cells) and typical WSSV histopathology (i.e. large basophilic, intranuclear viral inclusions in subcuticular and midgut epithelial cells) in shrimp (Fig. 5A–C), while these apoptotic signs and inclusions were not found in tissues of the shrimp administered rVP28-bs after WSSV challenge (Fig. 5D–F). In the pBS (control) group, after the WSSV challenge, multiple apoptotic bodies in intracellular and intercellular spaces as well as partly degraded apoptotic bodies were observed in hepatopancreas, subcuticular epithelium and midgut tissues (Fig. 5G–I). These apoptotic bodies did not appear in rVP28-bs group after challenge (Fig. 5J–L).

0.6

4. Discussion

0.4

As an attractive oral delivery vehicle, B. subtilis, a Gram-positive bacterium, has an efficient secretory mechanism that allows the presentation of foreign antigens to the host immunological system [26,27]. Our previous study has demonstrated that the recombinant

0.2 0.0 3

rVP28-e21

27

rVP28-bs

*

Bs

*

27

pBS

rVP28-bs

pBS

*

Caspase-3 activity (U)

iNOS activity (L-citrulline concentration µmol l-1)

C

14 28 Days post administration

18

9

18

9

0 3

14 28 Days post administration

Fig. 3. Phenoloxidase (PO) (A), Superoxide dismutase (SOD) (B) and inducible nitric oxide synthase (iNOS) (C) activities of F. chinensis after feeding rVP28-e21, rVP28-bs, B. subtilis (Bs) and pBS coated pellets at the 3rd, 14th and 28th day post-administration. The error bars indicate standard error of the mean (SEM). Statistical significance was marked by the star.

0

2

3

4

Days post challenge Fig. 4. Caspase-3 activities of rVP28-bs and pBS (control) groups on day 2, 3 and 4 post-challenge. The error bars indicate standard error of the mean (SEM).

54

L.-L. Fu et al. / Fish & Shellfish Immunology 28 (2010) 49–55

Fig. 5. Light microscope (LM) (magnification, 640) and transmission electron microscope (TEM) (magnification, scale bars) examination of experimental F. chinensis on day 2 post-challenge. Apoptotic bodies (a), cells of fragmented nuclei (*) and WSSV inclusions (arrow) are indicated.

B. subtilis strain in the form of spores, with the ability of high-level secretion of rVP28, can evoke protection of crayfish against WSSV [14]. It was also reported that oral administration of purified protein rVP28 expressed in Brevibacillus brevis resulted in the enhanced survival of Marsupenaeus japonicus against WSSV [28]. Thus, oral administration using a Gram-positive bacterium as expression vectors or delivery vehicles could be a potential strategy for prevention of disease in aquaculture. In the study presented here, we assessed the efficacy of oral administration of rVP28-bs in protection as well as immune responses of shrimp, F. chinensis, after challenge with WSSV. Challenge results showed that feeding rVP28-bs coated pellets conferred higher protection against WSSV than that of the rVP28e21 group. It is possible thereby to hypothesized that the presence of B. subtilis may by itself have a positive effect on shrimp survival upon WSSV challenge, which could also be deduced by the lower percentage of cumulative mortality of the Bs group compared with the positive control (Fig. 2). In an attempt to address the innate immune response of F. chinensis after oral administration with rVP28-bs, activities of the enzymes phenoloxidase (PO) and superoxide dismutase (SOD), were analyzed. As we know, PO, the key enzyme in the synthesis of melanin, can stimulate several humoral defense reactions in hemolymph [29]. Activated hemocytes also produce extra bactericidal

substances, such as H2O2 and superoxide anion (O2) that may increase disease resistance, and the toxic O2 can be rapidly catalyzed by SOD to molecular oxygen and hydrogen peroxide [30]. The results displayed that the supplementation of rVP28-bs into shrimp food pellets caused an increase of the activities of PO and SOD, which were higher than that in the rVP28-e21 group. Recently, Tseng et al. [20] demonstrated that shrimp fed the B. subtilis E20 exhibited significant increases in activities of PO, SOD, glutathione peroxidase etc. compared to control shrimp. All together, it was indicated that oral administration with rVP28-bs in F. chinensis stimulated protection against WSSV, as well as the innate immune system and these responses might be attributable, in part, to the probiotic activity of B. subtilis. Furthermore, the results showed that rVP28-bs and rVP28-e21 administration caused the more pronounced increase of iNOS activity compared to Bs, whereas the discrepancy of PO and SOD activities was not observed among these groups. It is well established that high levels of NO, produced by cytokines/endotoxininduced iNOS, play an important role in mammalian immune defense against tumor cell growth, bacterial, fungal, and viral infection [31]. Recent studies also confirmed that a Ca2þ-independent inducible NOS activity is present in hemocytes of shrimp [32] and in response to WSSV infection [12]. Thus, in this context,

L.-L. Fu et al. / Fish & Shellfish Immunology 28 (2010) 49–55

hemocyte-derived iNOS and NO appear to be involved in the innate immunity against virus infection in shrimp, while the proPO system and SOD clearance system play a comparatively minor role in antiviral defense of shrimp. However, the elucidation of the molecular mechanism is still in progress. By feeding rVP28-bs for 20 days, the most effective protection was available when shrimp were challenged at the 14th day postadministration. Meanwhile, the activities of PO, SOD and iNOS were also the highest at this time point. After immunization, the immune responses of F. chinensis seem to be stably maintained throughout this time course. These observations may contribute to formulate the standardization of immunization procedures to reduce WSSV infection in shrimp farming. It has been reported that, in WSSVinfected shrimp, apoptosis occurs followed by the increase of caspase-3 activity [11]. Besides this occurrence, we also revealed that after rVP28-bs administration in shrimp, the caspase-3 activity was suppressed and the signs of apoptosis (chromatin margination, nuclear fragmentation and apoptotic bodies) could not be observed, presumably by blocking WSSV infection. In conclusion, by oral delivery of VP28 using B. subtilis as a vehicle, shrimp (F. chinensis) showed significant resistance to WSSV, instead of the WSSV-caused death and apoptosis. The activity of iNOS was remarkably enhanced after feeding of rVP28-bs compared to that of PO and SOD, indicating that iNOS and NO may play an important role in antiviral defense of shrimp. Therefore, the results of this study could provide insights into the design of feasible prophylactic measures for diseases of shrimp using oral administration. Acknowledgements This work was supported by Science Fund for Young Scholars of Zhejiang Gongshang University, China (1110XJ130919) and the Natural Science Foundation of Zhejiang province, China (No. Y3090370). References [1] van Hulten MC, Witteveldt J, Peters S, Kloosterboer N, Tarchini R, Fiers M, et al. The white spot syndrome virus DNA genome sequence. Virology 2001;286: 7–22. [2] Mayo MA. A summary of taxonomic changes recently approved by ICTV. Arch Virol 2002;147:1655–6. [3] Zhang X, Huang C, Tang X, Zhuang Y, Hew CL. Identification of structural proteins from shrimp white spot syndrome virus (WSSV) by 2DE-MS. Proteins 2004;55:229–35. [4] Tsai JM, Wang HC, Leu JH, Hsiao HH, Wang AH, Kou GH, et al. Genomic and proteomic analysis of thirty-nine structural proteins of shrimp white spot syndrome virus. J Virol 2004;78:11360–70. [5] Xie X, Xu L, Yang F. Proteomic analysis of the major envelope and nucleocapsid proteins of white spot syndrome virus. J Virol 2006;80:10615–23. [6] Witteveldt J, Cifuentes CC, Vlak JM, van Hulten MC. Protection of Penaeus monodon against white spot syndrome virus by oral vaccination. J Virol 2004;78:2057–61. [7] Zhu F, Du H, Miao ZG, Quan HZ, Xu ZR. Protection of Procambarus clarkii against white spot syndrome virus using inactivated WSSV. Fish Shellfish Immunol 2009;26:685–90. [8] Sarathi M, Ahmed V, Venkatesan C, Balasubramanian G, Prabavathy J, Hameed A. Comparative study on immune response of Fenneropenaeus indicus to Vibrio alginolyticus and white spot syndrome virus. Aquaculture 2007;271: 8–20.

55

[9] Amparyup P, Charoensapsri W, Tassanakajon A. Two prophenoloxidases are important for the survival of Vibrio harveyi challenged shrimp Penaeus monodon. Dev Comp Immunol 2009;33:247–56. [10] Li CC, Yeh ST, Chen JC. The immune response of white shrimp Litopenaeus vannamei following Vibrio alginolyticus injection. Fish Shellfish Immunol 2008;25:853–60. [11] Wongprasert K, Khanobdee K, Glunukarn SS, Meeratana P, Withyachumnarnkul B. Time-course and levels of apoptosis in various tissues of black tiger shrimp Penaeus monodon infected with white spot syndrome virus. Dis Aquat Org 2003;55:3–10. [12] Jiang G, Yu R, Zhou M. Studies on nitric oxide synthase activity in haemocytes of shrimps Fenneropenaeus chinensis and Marsupenaeus japonicus after white spot syndrome virus infection. Nitric Oxide 2006;14:219–27. [13] Namikoshi A, Wu JL, Yamashita T, Nishizawa T, Nishioka T, Arimoto M, et al. Vaccination trials with Penaeus japonicus to induce resistance to white spot syndrome virus. Aquaculture 2004;229:25–35. [14] Fu LL, Li WF, Du HH, Dai W, Xu ZR. Oral vaccination with envelope protein VP28 against white spot syndrome virus in Procambarus clarkii using Bacillus subtilis as delivery vehicles. Lett Appl Microbiol 2008;46:581–6. [15] van Hulten MCW, Witteveldt J, Snippe M, Vlak JM. White spot syndrome virus envelope protein VP28 is involved in the systemic infection of shrimp. Virology 2001;285:228–33. [16] Yi G, Wang Z, Qi Y, Yao L, Qian J, Hu L. VP28 of shrimp white spot syndrome virus is involved in the attachment and penetration into shrimp cells. J Biochem Mol Biol 2004;37:726–34. [17] Sritunyalucksana K, Wannapapho W, Lo CF, Flegel TW. PmRab7 is a VP28binding protein involved in white spot syndrome virus infection in shrimp. J Virol 2006;80:10734–42. [18] Du HH, Xu ZR, Wu XF, Li WF, Dai W. Increased resistance to white spot syndrome virus in Procambarus clarkii by injection of envelope protein VP28 expressed using recombinant baculovirus. Aquaculture 2006;260:39–43. [19] Jha RK, Xu ZR, Bai SJ, Sun JY, Li WF, Shen J. Protection of Procambarus clarkia against white spot syndrome virus using vaccine expressed in Pichia pastoris. Fish Shellfish Immunol 2006;22:295–307. [20] Tseng DY, Ho PL, Huang SY, Cheng SC, Shiu YL, Chiu CS, et al. Enhancement of immunity and disease resistance in the white shrimp, Litopenaeus vannamei, by the probiotic, Bacillus subtilis E20. Fish Shellfish Immunol 2009;26:339–44. [21] Nicholson WL, Setlow P. Sporulation, germination and outgrowth. In: Harwood CR, Cutting SM, editors. Molecular biological methods for Bacillus. Chichester: John Wiley & Sons Ltd; 1990. p. 391–450. [22] Du HH, Fu LL, Xu YX, Kil ZS, Xu ZR. Improvement in a simple method for isolating white spot syndrome virus (WSSV) from the crayfish Procambarus clarkii. Aquaculture 2007;262:532–4. [23] Vaseeharan B, Jayakumar R, Ramasamy P. PCR-based detection of white spot syndrome virus in cultured and captured crustaceans in India. Lett Appl Microbiol 2003;37:443–7. ˜ andez-Lo´pez J, Gollas-Galva´n T, Vargas-Albores F. Activation of the [24] Hern prophenoloxidase system of the brown shrimp (Penaeus californensis Holmes). Comp Biochem Physiol 1996;113:61–6. [25] Amend DF. Potency testing of fish vaccines. In: Anderson DP, Hennessen W, editors. Fish Biologics: serodiagnostics and vaccines. Basel: S Karger; 1981. p. 447–54. [26] Hoang TH, Hong HA, Clark GC, Titball RW, Cutting SM. Recombinant Bacillus subtilis expressing the Clostridium perfringens alpha toxoid is a candidate orally delivered vaccine against necrotic enteritis. Infect Immun 2008;76:5257–65. [27] Paccez JD, Nguyen HD, Luiz WB, Ferreira RC, Sbrogio-Almeida ME, Schuman W, et al. Evaluation of different promoter sequences and antigen sorting signals on the immunogenicity of Bacillus subtilis vaccine vehicles. Vaccine 2007;25:4671–80. [28] Caipang CM, Verjan N, Ooi EL, Kondo H, Hirono I, Aoki T, et al. Enhanced survival of shrimp, Penaeus (Marsupenaeus) japonicus from white spot syndrome disease after oral administration of recombinant VP28 expressed in Brevibacillus brevis. Fish Shellfish Immunol 2008;25:315–20. [29] Wang W, Zhang X. Comparison of antiviral efficiency of immune responses in shrimp. Fish Shellfish Immunol 2008;25:522–7. [30] Song YL, Hsieh YT. Immunostimulation of tiger shrimp Penaeus monodon haemocytes for generation of microbicidal substances: analysis of reactive oxygen species. Dev Comp Immunol 1994;18:201–9. [31] Whittle BJ. Nitric oxide in physiology and pathology. Histochem J 1995;27: 727–37. [32] Yeh FC, Wu SH, Lai CY, Lee CY. Demonstration of nitric oxide synthase activity in crustacean hemocytes and anti-microbial activity of hemocyte-derived nitric oxide. Comp Biochem Physiol B Biochem Mol Biol 2006;144:11–7.