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In vivo assessment for oral delivery of Bacillus subtilis harboring a viral protein (VP28) against white spot syndrome virus in Litopenaeus vannamei
Aquaculture 322-323 (2011) 33–38
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In vivo assessment for oral delivery of Bacillus subtilis harboring a viral protein (VP28) against white spot syndrome virus in Litopenaeus vannamei Ling-Lin Fu a,⁎, Yanbo Wang a, Zheng-Cun Wu a, Wei-Fen Li b,⁎⁎ a b
Food Safety Key Laboratory of Zhejiang Province, School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, 310035, P.R. China Key Laboratory of Molecular Animal Nutrition, Ministry of Education, College of Animal Sciences, Zhejiang University, Hangzhou, 310058, P. R. China
a r t i c l e
i n f o
Article history: Received 24 August 2011 Received in revised form 21 September 2011 Accepted 27 September 2011 Available online 2 October 2011 Keywords: White spot syndrome virus Litopenaeus vannamei rVP28-bs Oral delivery Phagocytosis Dissemination
a b s t r a c t Bacillus subtilis spores, being consumed as probiotics, have been explored as live carriers for expression and oral delivery of antigen proteins. In our initial experiment, by oral delivery of B. subtilis spores harboring VP28 (rVP28-bs) to Litopenaeus vannamei, the extremely high survival (Relative Percent Survival: 83.3%) upon challenge with white spot syndrome virus (WSSV) can be observed. After ‘vaccination’ with rVP28-bs, the hemocytic phagocytosis and immune-related gene expression levels in hemocytes were analyzed. The percentage of haemocytes phagocytosing WSSV was significantly higher (p b 0.001) in shrimp previously fed with rVP28-bs (48.2 ± 6.3) than the controls (11.0 ± 3.5 and 8.1 ± 2.5). However, there were no significant differences (p N 0.05) in all the experimental groups for the percentage phagocytosis of TSV (an unrelated virus of shrimp). This suggests that the heightened phagocytic activity, and thus the high-level survival of shrimp after rVP28-bs ‘vaccination’ are selective or specific towards WSSV. Moreover, immune-related genes (proPO, PE and LGBP) were significantly (p b 0.05) upregulated in both rVP28-bs and B. subtilis feeding groups compared to the control, though no significant differences (p N 0.05) were observed between rVP28bs and B. subtilis groups. It was indicated that the phagocytosis enhanced by rVP28-bs was the essential one to protect shrimp from virus infection, while the rVP28-bs-stimulated humoral response only plays an assistant role in antiviral defense of shrimp. Besides, in vivo fate and dissemination assays of rVP28-bs spores showed that, as robust life forms, spores can survive transit across the gut tract, germinate to express rVP28 and exert probiotic action, and disseminate in the haemolymph to present VP28 to the shrimp defense system before being excreted. These results may raise the application prospective of this recombinant B. subtilis spores against WSSV infection in shrimp farms. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The shrimp culture industry has been severely hampered by outbreaks of white spot syndrome virus (WSSV) worldwide (Chang et al., 1998). Although considerable progress has been made in WSSV molecular characterization (Tsai et al., 2004; Xie et al., 2006; Zhang et al., 2004) as well as potential anti-WSSV strategies based on shrimp defense system (Witteveldt et al., 2004; Zhu et al., 2009), no adequate treatment is available. It was mostly attributed to two obstacles: (i) the poor understanding of WSSV infection and replication mechanisms, and (ii) the molecular process of shrimp immune responses against this virus.
⁎ Correspondence to: L.-L. Fu, Food Safety Key Laboratory of Zhejiang Province, School of Food Science and Biotechnology, Zhejiang Gongshang University, 149 Jiao Gong Road, Hangzhou, 310035, P.R. China. Tel.: + 86 571 88071024 7589. ⁎⁎ Correspondence to: W.-F. Li, Key Laboratory of Molecular Animal Nutrition, Ministry of Education, College of Animal Sciences, Zhejiang University, 866 Yu Hang Tang Road, Hangzhou, 310058, P. R. China. Tel.: +86 571 88982108; fax: +86 571 88982117. E-mail addresses: [email protected] (L.-L. Fu), wfl[email protected] (W.-F. Li). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.09.036
Generally, crustaceans are considered to lack a true adaptive immune system, but efficient to protect and preserve themselves from all intruding pathogens based on the innate immune system (Sarathi et al., 2007). Recent reports, however, have suggested the existence of some form of immune memory in invertebrates that is referred to by some authors as ‘immune priming’ or ‘specific immune priming’ (Johnson et al., 2008; Litman et al., 2007; Rowley and Powell, 2007). It is also reported that memory responses could be inducible in shrimp using either inactivated pathogens or recombinant proteins against WSSV (Fu et al., 2008; Namikoshi et al., 2004; Witteveldt et al., 2004; Zhu et al., 2009). The existence of specific immune priming in shrimp makes ‘vaccination’ desirable and feasible. VP28, one of the major envelope proteins of WSSV, has been shown to be a potential WSSV ‘vaccine’ candidate (Du et al., 2006; Sritunyalucksana et al., 2006; van Hulten et al., 2001; Witteveldt et al., 2004; Yi et al., 2004). In previous papers, we reported that the recombinant Bacillus subtilis strain with the ability of high-level secretion of rVP28 can evoke protection of crayfish (Fu et al., 2008) and Fenneropenaeus chinensis (Fu et al., 2010) against WSSV by oral delivery. B. subtilis, the well-known host for industrial enzyme production, has
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been explored as a tool for expression and delivery of antigen proteins, due to the immense capacity of secreting proteins, lack of pathogenicity, robust life form spores and function of immune stimulation (Fu et al., 2008; Tseng et al., 2009). However, whether the heightened resistance caused by this recombinant B. subtilis strain in shrimp was specific against WSSV infection still remains to be confirmed. The fate and dissemination of this strain in shrimp after oral delivery also needs to be studied for the further application. Therefore, this study examines whether fed with the B. subtilis spores harboring VP28 can result in specifically heightened cellular (phagocytosis) and humoral (proPO, PE and LGBP gene expression) immune responses against WSSV in Pacific white shrimp (Litopenaeus vannamei). Moreover, we have addressed the question of what happens to spores taken orally in shrimp. 2. Materials and methods 2.1. Materials 2.1.1. Recombinant strains As we reported, the vp28 gene was introduced into B. subtilis WB600 to form the recombinant strain rVP28-bs which can secrete rVP28 at a high level (Fu et al., 2008). Another recombinant B. subtilis WB600 strain harboring the green fluorescent protein (GFP) and VP28 fusion protein (rVP28-bs-gfp) was also constructed and stored in our laboratory (unpublished) for the dissemination assay in this study. 2.1.2. Coating of feed pellets Purification of B. subtilis spores was conducted in DSM (Difcosporulation media) using the exhaustion method (Nicholson and Setlow, 1990). The rVP28-bs and rVP28-bs-gfp suspension containing 10 8 spores was coated with 0.01 g of commercial pellets (Crown Feed Co. Ltd., China), respectively, as previously described (Fu et al., 2010). The B. subtilis WB600 strain was also coated for the control group. The prepared food pellets were stored at 4 °C until further use. 2.1.3. Virus The WSSV stocks were generated and stored in our laboratory ((Du et al., 2007)) at −80 °C. The purified virions of Taura syndrome virus (TSV) were kindly supplied by Dr. Shuai Jiang-Bing (Zhejiang Entry & Exit Inspection and Quarantine Bureau, Hangzhou, China). 2.2. Shrimp culture Pacific white shrimp (L. vannamei), 10–15 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 sand-filtered, ozone-treated and flow-through salt water (30‰) at 22–25 °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 before and during the experiments.
EDTA; pH 4.6) over ice for the subsequent immune-related gene expression and phagocytosis assays. On the 7th day after 20-day feeding, the remaining shrimp were challenged with WSSV or pBS buffer. Before the beginning of the experiments, in vivo titration of WSSV was conducted as previously described (Fu et al., 2010). In order to mimic the natural route of infection and the initial situation in a pond, shrimp were challenged via immersion. Dead shrimp were tested for the presence of WSSV by PCR (Vaseeharan et al., 2003). Mortality was recorded for 25 days post-challenge. In Experiment 2, shrimp were divided into two groups designated as A and B, and fed with food pellets totally containing 2.75 × 10 10 spores of rVP28-bs-gfp to study in vivo fate and dissemination. For group A, gut contents were collected at various time points (6, 12, 24, 72, 144 h) after feeding and processed immediately to determine the spore counts. For group B, shrimp were sacrificed at 1, 3, 7 and 14 days after feeding and dissected for selected organs and tissues (gut, hepatopancreas, gill, stomach, heart, haemolymph and muscle). The specimens were then processed for immunofluorescence staining and gfp gene detection to demonstrate in vivo dissemination. The primers for PCR detection of gfp gene harbored in rVP28bs-gfp were BS-gfp-F (5’-GTAAG AGAGG AATGT ACACA T-3’) and BS-gfp-R (CCGGA ATTCC TATTT GTATA GT-3’). 2.4. Real-time PCR quantification of immune-related gene expressions Hemocytes were collected by centrifugation according to the method of a previous study (Liu et al., 2005). Total RNA of hemocytes was isolated using the guanidinium thiocyanate method described by Chomczynski and Sacchi (1987). The real-time PCR quantification of proPO, LGBP and PE gene expressions in hemocytes was based on the method of Yeh et al. (2009). 2.5. Fluorescent labeling of virus and phagocytosis assay The WSSV and TSV virions were incubated in 0.1 M NaHCO3 containing 1 mg/mL FITC isomer 1 (Sigma) for fluorescent labeling following the method described by Wu et al. (2008). The phagocytosis assay was carried out by the method as described (Pope et al., 2011). The phagocytic percentage (PP) was calculated with the equation PP= (number of cells ingesting virus/number of cells observed) × 100%. 2.6. Spore counts To determine the number of spores in gut contents, samples were suspended in 10 ml of phosphate-buffered saline (PBS) at 65 °C. Sterile glass beads (2 mm, 3 ml) were added, and the suspension was incubated at 65 °C for 1 h with frequent vortexing until there was little remaining residual solid matter. Serial dilutions were then made with PBS (65 °C), plated on DSM plates containing kanamycin (1 mg/ml) and Ampicillin (5 mg/ml), and incubated at 37 °C for 2 days. B. subtilis colonies of strain rVP28-bs-gfp were identified by their colony morphology. Spore counts were extrapolated for the total weight of gut contents collected.
2.3. Experimental design 2.7. Immunofluorescence staining In Experiment 1, shrimp were divided into three groups, and each group with two subgroups. There were 20 shrimp in triplicate in each subgroup. The three groups were treated by feeding rVP28bs-, B. subtilis- and pBS buffer-coated food pellets, respectively, for 20 days. At the end of oral delivery, part of shrimp were anaesthetised by placing on ice and pierced to collect haemolymph using a wide-bore (19 G) needle. The haemolymph was collected in a Petridish containing 5 ml ice-cold marine anticoagulant (0.45 M NaCl, 0.1 M glucose, 30 mM trisodium citrate, 26 mM citric acid, 10 mM
The immunofluorescence staining of tissue specimens was based on the method that described previously (Diehlmann et al., 2011) with some modifications. For rVP28-bs-gfp detection, a mouse monoclonal anti-gfp (AbCam, Cambridge, United Kingdom) was used in conjunction with a FITC- labeled secondary anti-mouse antibody (Invitrogen, Carlsbad, CA). Propidium Iodide (KeyGEN Biotech, Nanjing, China) was used for nuclear staining. Specimens were viewed and analyzed using a TSC SP5 laser scanning confocal microscope (Leica, Germany).
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Statistical calculations were performed using SPSS (version 9.0) software. Significant difference was indicated by one-way ANOVA (pb 0.05) analysis. The protection against WSSV after administration was calculated as the relative percent survival (RPS) (Amend, 1981): oral delivered group mortality ×100 RPS ¼ 1− control group mortality
3. Results
LGBP mRNA -1 -6 (10 ng ¦Ìg total RNA )
A
2.8. Statistical analysis
3.1. Increased survival of L. vannamei against WSSV by rVP28-bs delivery
The expressions of the proPO, LGBP and PE genes were significantly higher (p b 0.05) in both rVP28-bs and B. subtilis groups compared to the control (oral delivered by pBS coating feed pellets) (Fig. 2). However, no significant differences in proPO, LGBP and PE mRNA transcription levels of shrimp were observed between rVP28-bs and B. subtilis groups. 3.3. Specific hemocytic phagocytosis induced by rVP28-bs delivery Shrimp previously fed with rVP28-bs showed significantly more fluorescently-labelled WSSV in haemocytes (blood cells) than those fed with B. subtilis and pBS coating feed pellets (Fig. 3A). Similarly, the percentage of haemocytes phagocytosing WSSV was significantly higher (p b 0.001) in shrimp previously fed with rVP28-bs (48.2± 6.3) than those fed with B. subtilis (11.0 ± 3.5) and pBS (8.1 ± 2.5) coating
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B proPO mRNA -1 ¦Ìg total RNA ) (10
3.2. Effect of oral delivery on immune-related gene expressions
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PE mRNA -1 -3 (10 ng ¦Ìg total RNA )
In Experiment 1, oral delivery of rVP28-bs spores harboring the WSSV envelope protein VP28 resulted in a significantly lower cumulative mortality (p b 0.001) of L. vannamei at the challenge point of 7 days after 20-day feeding (Fig. 1). The calculated RPS value of rVP28-bs group was 83.3% compared with the positive control. Moreover, by oral delivery of B. subtilis spores, the number of survivors was also slightly increased after WSSV challenge leading to RPS value of 21.7% compared with the positive control. The negative control showed no mortality. Three randomly selected survivors from each group were tested for WSSV by PCR and tested negative.
35
rVP28-bs
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90 Fig. 2. Analysis of prophenoloxidase (proPO) (A), peroxinectin (PE) (B) and lipopolysaccharide and β-1,3-glucan binding protein (LGBP) (C) gene expression in hemocytes of the experimental groups by SYBR green real-time RT-PCR. Mean (± SEM) with different letters were significantly different (p b 0.05) among treatments.
80 70 60 50 40
feed pellets (Fig. 3B). There were no significant differences between B. subtilis and pBS groups for the phagocytic percentage of WSSV. Finally, there were no significant differences in all groups for the percentage phagocytosis of TSV (an unrelated pathogenic virus of shrimp) (Fig. 3).
30 20 10 0 0
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Fig. 1. Protective effects against WSSV of recombinant VP28 by oral delivery of spores of rVP28-bs strain to L. vannamei. Cumulative mortality rates of shrimp from the experimental groups were indicated. Shrimp were challenged 7 days after cessation of feeding coated pellets. The error bars indicate standard error of the mean (±SEM). Statistical significance was marked by the star.
3.4. Fate and dissemination of recombinant strain spores in L. vannamei In Experiment 2, shrimp were fed with 2.75 × 1010 spores in total, and measured the number of spores excreted. Our results showed that the majority of counts occurred within the first 24 h (4.0 × 1010), although spores were still detectable in the 6-day samples (7.2× 102). The cumulative number of spores excreted was 4.78 × 1010, and thus the ratio of spore counts to inoculum was 1.74. Moreover, we found that oral delivery produced relatively high levels of recombinant spore dissemination in the shrimp gut and stomach at 1, 3 and 7 days after
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A
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TSV
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Fig. 3. Evaluation of specificity of phagocytosis towards WSSV in L. vannamei hemocytes following feeding of rVP28-bs strain. Phagocytosing an unrelated virus (TSV) was also conducted as the control. (A) Hemocytes from rVP28-bs, B. subtilis and pBS groups phagocytosing FITC-labeled WSSV and TSV were examined under fluorescent microscope. (B) The percentage of haemocytes that showed phagocytic activity was recorded. The error bars indicate standard error of the mean (±SEM). Statistical significance was marked by the star.
feeding. The spores could also be detected at low levels in hepatopancreas at 3 days after feeding as well as in haemolymph at 3 and 7 days after feeding. It should be noted that spores cannot be detected at 14 days after feeding in all the above tissues (Fig. 4). Finally, no detectable spores appeared in the gill, heart and muscle of shrimp (data not shown). 4. Discussion In this study, challenge result showed that feeding rVP28-bs coated pellets conferred significantly higher protection of L. vannamei against WSSV (RPS: 83.3%) than that of the positive control, which was consistent with our previous results carried out in Procambarus clarkii (Fu et al., 2008) and F. chinensis (Fu et al., 2010). Furthermore, we have demonstrated that previous feeding of rVP28-bs spores to L. vannamei results in the increased phagocytic uptake of WSSV by the haemocytes. The phagocytosis of an unrelated virus of shrimp (TSV) by the same haemocytes was insignificantly changed compared to cells taken from shrimp fed with pBS coating feed. This suggests the stimulation of phagocytic activity, and thus leading to the high-level protection in shrimp after rVP28-bs ‘vaccination’ is selective or specific towards WSSV. Our results agree with those of another study using L. vannamei that observed enhanced phagocytosis of the bacterial shrimp pathogen, Vibrio harveyi, after challenge with the same species (Pope et al., 2011). To date, Dscam is one of the best candidates to explain the phenomenon of specific immune priming in invertebrate including shrimp (Chou et al., 2009, 2011; Dong et al., 2006; Watson et al., 2005). The mechanistic explanations for how the immune system accomplishes such heightened resistance to infection could arise the attention to the development of putative ‘vaccines’ aimed to boost growth and welfare in shrimp farming. Another observation of the current study is that humoral immunerelated genes (proPO, PE and LGBP) were significantly upregulated in both rVP28-bs and B. subtilis feeding groups compared to the control.
As an oral ‘vaccine’ carrier, B. subtilis might act as an adjuvant in the ‘vaccination’ leading to the enhanced humoral immune response in shrimp. However, the extremely high survival of L. vannamei challenged by WSSV could only be observed in the rVP28-bs group that also demonstrated a significantly high percentage of haemocytes phagocytosing WSSV. Taken together, it was indicated that the cellular response stimulated by rVP28-bs was the essential one to protect shrimp from virus infection, while the heightened humoral response only plays an assistant role in antiviral defense of shrimp. In Experiment 2, the total accumulated number of spores excreted over 144 h was higher than the original inoculum. Moreover, considerable numbers of spores were still being detected in the gut contents on day 6, and there was a gradual decline in spore counts over time. Our interpretation is that a proportion of the spores had germinated in the gut of shrimp, undergone one or more rounds of growth and replication, and then formed spores, which was similar to the results stated by Hoa et al. (2001) in a murine model. Thus, B. subtilis possibly exert their probiotic action (such as stimulation of humoral immune response) by a metabolic effect following spore germination. Another intriguing observation was that the B. subtilis spores harboring VP28 could be detected in the shrimp haemolymph after feeding. The dissemination of this live carrier in haemolymph by oral delivery might facilitate to present the antigen protein to the host defense system when secretedly express VP28. In conclusion, the heightened phagocytic activity, and thus the high-level survival of shrimp after rVP28-bs ‘vaccination’ are selective or specific towards WSSV. The phagocytosis enhanced by rVP28-bs was the essential one to protect shrimp from virus infection, while the heightened humoral response only plays an assistant role in antiviral defense of shrimp. Furthermore, as robust life forms, spores can survive transit across the gastrointestinal tract, germinate and disseminate in the haemolymph to present the antigen protein VP28 to host defense system before being excreted.
L.-L. Fu et al. / Aquaculture 322-323 (2011) 33–38
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Fig. 4. Spore dissemination examined by PCR detection (A) and immunofluorescence staining (B). Shrimp were sacrificed at 1, 3, 7 and 14 days after feeding and dissected for selected organs and tissues (gut, hepatopancreas, gill, stomach, heart, haemolymph and muscle) to detect in vivo dissemination. No detectable spores appeared in the gill, heart and muscle of shrimp (data not shown).
Acknowledgements This study was financially supported by the National Natural Science Foundation of China (31101921), the Zhejiang Provincial Natural Science Foundation of China (Y3090370 and R3110345), and the Science and Technology Development Plan of Hangzhou, China (20101032B47).
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In vivo assessment for oral delivery of Bacillus subtilis harboring a viral protein (VP28) against white spot syndrome virus in Litopenaeus vannamei Ling-Lin Fu a,⁎, Yanbo Wang a, Zheng-Cun Wu a, Wei-Fen Li b,⁎⁎ a b
Food Safety Key Laboratory of Zhejiang Province, School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, 310035, P.R. China Key Laboratory of Molecular Animal Nutrition, Ministry of Education, College of Animal Sciences, Zhejiang University, Hangzhou, 310058, P. R. China
a r t i c l e
i n f o
Article history: Received 24 August 2011 Received in revised form 21 September 2011 Accepted 27 September 2011 Available online 2 October 2011 Keywords: White spot syndrome virus Litopenaeus vannamei rVP28-bs Oral delivery Phagocytosis Dissemination
a b s t r a c t Bacillus subtilis spores, being consumed as probiotics, have been explored as live carriers for expression and oral delivery of antigen proteins. In our initial experiment, by oral delivery of B. subtilis spores harboring VP28 (rVP28-bs) to Litopenaeus vannamei, the extremely high survival (Relative Percent Survival: 83.3%) upon challenge with white spot syndrome virus (WSSV) can be observed. After ‘vaccination’ with rVP28-bs, the hemocytic phagocytosis and immune-related gene expression levels in hemocytes were analyzed. The percentage of haemocytes phagocytosing WSSV was significantly higher (p b 0.001) in shrimp previously fed with rVP28-bs (48.2 ± 6.3) than the controls (11.0 ± 3.5 and 8.1 ± 2.5). However, there were no significant differences (p N 0.05) in all the experimental groups for the percentage phagocytosis of TSV (an unrelated virus of shrimp). This suggests that the heightened phagocytic activity, and thus the high-level survival of shrimp after rVP28-bs ‘vaccination’ are selective or specific towards WSSV. Moreover, immune-related genes (proPO, PE and LGBP) were significantly (p b 0.05) upregulated in both rVP28-bs and B. subtilis feeding groups compared to the control, though no significant differences (p N 0.05) were observed between rVP28bs and B. subtilis groups. It was indicated that the phagocytosis enhanced by rVP28-bs was the essential one to protect shrimp from virus infection, while the rVP28-bs-stimulated humoral response only plays an assistant role in antiviral defense of shrimp. Besides, in vivo fate and dissemination assays of rVP28-bs spores showed that, as robust life forms, spores can survive transit across the gut tract, germinate to express rVP28 and exert probiotic action, and disseminate in the haemolymph to present VP28 to the shrimp defense system before being excreted. These results may raise the application prospective of this recombinant B. subtilis spores against WSSV infection in shrimp farms. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The shrimp culture industry has been severely hampered by outbreaks of white spot syndrome virus (WSSV) worldwide (Chang et al., 1998). Although considerable progress has been made in WSSV molecular characterization (Tsai et al., 2004; Xie et al., 2006; Zhang et al., 2004) as well as potential anti-WSSV strategies based on shrimp defense system (Witteveldt et al., 2004; Zhu et al., 2009), no adequate treatment is available. It was mostly attributed to two obstacles: (i) the poor understanding of WSSV infection and replication mechanisms, and (ii) the molecular process of shrimp immune responses against this virus.
⁎ Correspondence to: L.-L. Fu, Food Safety Key Laboratory of Zhejiang Province, School of Food Science and Biotechnology, Zhejiang Gongshang University, 149 Jiao Gong Road, Hangzhou, 310035, P.R. China. Tel.: + 86 571 88071024 7589. ⁎⁎ Correspondence to: W.-F. Li, Key Laboratory of Molecular Animal Nutrition, Ministry of Education, College of Animal Sciences, Zhejiang University, 866 Yu Hang Tang Road, Hangzhou, 310058, P. R. China. Tel.: +86 571 88982108; fax: +86 571 88982117. E-mail addresses: [email protected] (L.-L. Fu), wfl[email protected] (W.-F. Li). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.09.036
Generally, crustaceans are considered to lack a true adaptive immune system, but efficient to protect and preserve themselves from all intruding pathogens based on the innate immune system (Sarathi et al., 2007). Recent reports, however, have suggested the existence of some form of immune memory in invertebrates that is referred to by some authors as ‘immune priming’ or ‘specific immune priming’ (Johnson et al., 2008; Litman et al., 2007; Rowley and Powell, 2007). It is also reported that memory responses could be inducible in shrimp using either inactivated pathogens or recombinant proteins against WSSV (Fu et al., 2008; Namikoshi et al., 2004; Witteveldt et al., 2004; Zhu et al., 2009). The existence of specific immune priming in shrimp makes ‘vaccination’ desirable and feasible. VP28, one of the major envelope proteins of WSSV, has been shown to be a potential WSSV ‘vaccine’ candidate (Du et al., 2006; Sritunyalucksana et al., 2006; van Hulten et al., 2001; Witteveldt et al., 2004; Yi et al., 2004). In previous papers, we reported that the recombinant Bacillus subtilis strain with the ability of high-level secretion of rVP28 can evoke protection of crayfish (Fu et al., 2008) and Fenneropenaeus chinensis (Fu et al., 2010) against WSSV by oral delivery. B. subtilis, the well-known host for industrial enzyme production, has
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been explored as a tool for expression and delivery of antigen proteins, due to the immense capacity of secreting proteins, lack of pathogenicity, robust life form spores and function of immune stimulation (Fu et al., 2008; Tseng et al., 2009). However, whether the heightened resistance caused by this recombinant B. subtilis strain in shrimp was specific against WSSV infection still remains to be confirmed. The fate and dissemination of this strain in shrimp after oral delivery also needs to be studied for the further application. Therefore, this study examines whether fed with the B. subtilis spores harboring VP28 can result in specifically heightened cellular (phagocytosis) and humoral (proPO, PE and LGBP gene expression) immune responses against WSSV in Pacific white shrimp (Litopenaeus vannamei). Moreover, we have addressed the question of what happens to spores taken orally in shrimp. 2. Materials and methods 2.1. Materials 2.1.1. Recombinant strains As we reported, the vp28 gene was introduced into B. subtilis WB600 to form the recombinant strain rVP28-bs which can secrete rVP28 at a high level (Fu et al., 2008). Another recombinant B. subtilis WB600 strain harboring the green fluorescent protein (GFP) and VP28 fusion protein (rVP28-bs-gfp) was also constructed and stored in our laboratory (unpublished) for the dissemination assay in this study. 2.1.2. Coating of feed pellets Purification of B. subtilis spores was conducted in DSM (Difcosporulation media) using the exhaustion method (Nicholson and Setlow, 1990). The rVP28-bs and rVP28-bs-gfp suspension containing 10 8 spores was coated with 0.01 g of commercial pellets (Crown Feed Co. Ltd., China), respectively, as previously described (Fu et al., 2010). The B. subtilis WB600 strain was also coated for the control group. The prepared food pellets were stored at 4 °C until further use. 2.1.3. Virus The WSSV stocks were generated and stored in our laboratory ((Du et al., 2007)) at −80 °C. The purified virions of Taura syndrome virus (TSV) were kindly supplied by Dr. Shuai Jiang-Bing (Zhejiang Entry & Exit Inspection and Quarantine Bureau, Hangzhou, China). 2.2. Shrimp culture Pacific white shrimp (L. vannamei), 10–15 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 sand-filtered, ozone-treated and flow-through salt water (30‰) at 22–25 °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 before and during the experiments.
EDTA; pH 4.6) over ice for the subsequent immune-related gene expression and phagocytosis assays. On the 7th day after 20-day feeding, the remaining shrimp were challenged with WSSV or pBS buffer. Before the beginning of the experiments, in vivo titration of WSSV was conducted as previously described (Fu et al., 2010). In order to mimic the natural route of infection and the initial situation in a pond, shrimp were challenged via immersion. Dead shrimp were tested for the presence of WSSV by PCR (Vaseeharan et al., 2003). Mortality was recorded for 25 days post-challenge. In Experiment 2, shrimp were divided into two groups designated as A and B, and fed with food pellets totally containing 2.75 × 10 10 spores of rVP28-bs-gfp to study in vivo fate and dissemination. For group A, gut contents were collected at various time points (6, 12, 24, 72, 144 h) after feeding and processed immediately to determine the spore counts. For group B, shrimp were sacrificed at 1, 3, 7 and 14 days after feeding and dissected for selected organs and tissues (gut, hepatopancreas, gill, stomach, heart, haemolymph and muscle). The specimens were then processed for immunofluorescence staining and gfp gene detection to demonstrate in vivo dissemination. The primers for PCR detection of gfp gene harbored in rVP28bs-gfp were BS-gfp-F (5’-GTAAG AGAGG AATGT ACACA T-3’) and BS-gfp-R (CCGGA ATTCC TATTT GTATA GT-3’). 2.4. Real-time PCR quantification of immune-related gene expressions Hemocytes were collected by centrifugation according to the method of a previous study (Liu et al., 2005). Total RNA of hemocytes was isolated using the guanidinium thiocyanate method described by Chomczynski and Sacchi (1987). The real-time PCR quantification of proPO, LGBP and PE gene expressions in hemocytes was based on the method of Yeh et al. (2009). 2.5. Fluorescent labeling of virus and phagocytosis assay The WSSV and TSV virions were incubated in 0.1 M NaHCO3 containing 1 mg/mL FITC isomer 1 (Sigma) for fluorescent labeling following the method described by Wu et al. (2008). The phagocytosis assay was carried out by the method as described (Pope et al., 2011). The phagocytic percentage (PP) was calculated with the equation PP= (number of cells ingesting virus/number of cells observed) × 100%. 2.6. Spore counts To determine the number of spores in gut contents, samples were suspended in 10 ml of phosphate-buffered saline (PBS) at 65 °C. Sterile glass beads (2 mm, 3 ml) were added, and the suspension was incubated at 65 °C for 1 h with frequent vortexing until there was little remaining residual solid matter. Serial dilutions were then made with PBS (65 °C), plated on DSM plates containing kanamycin (1 mg/ml) and Ampicillin (5 mg/ml), and incubated at 37 °C for 2 days. B. subtilis colonies of strain rVP28-bs-gfp were identified by their colony morphology. Spore counts were extrapolated for the total weight of gut contents collected.
2.3. Experimental design 2.7. Immunofluorescence staining In Experiment 1, shrimp were divided into three groups, and each group with two subgroups. There were 20 shrimp in triplicate in each subgroup. The three groups were treated by feeding rVP28bs-, B. subtilis- and pBS buffer-coated food pellets, respectively, for 20 days. At the end of oral delivery, part of shrimp were anaesthetised by placing on ice and pierced to collect haemolymph using a wide-bore (19 G) needle. The haemolymph was collected in a Petridish containing 5 ml ice-cold marine anticoagulant (0.45 M NaCl, 0.1 M glucose, 30 mM trisodium citrate, 26 mM citric acid, 10 mM
The immunofluorescence staining of tissue specimens was based on the method that described previously (Diehlmann et al., 2011) with some modifications. For rVP28-bs-gfp detection, a mouse monoclonal anti-gfp (AbCam, Cambridge, United Kingdom) was used in conjunction with a FITC- labeled secondary anti-mouse antibody (Invitrogen, Carlsbad, CA). Propidium Iodide (KeyGEN Biotech, Nanjing, China) was used for nuclear staining. Specimens were viewed and analyzed using a TSC SP5 laser scanning confocal microscope (Leica, Germany).
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Statistical calculations were performed using SPSS (version 9.0) software. Significant difference was indicated by one-way ANOVA (pb 0.05) analysis. The protection against WSSV after administration was calculated as the relative percent survival (RPS) (Amend, 1981): oral delivered group mortality ×100 RPS ¼ 1− control group mortality
3. Results
LGBP mRNA -1 -6 (10 ng ¦Ìg total RNA )
A
2.8. Statistical analysis
3.1. Increased survival of L. vannamei against WSSV by rVP28-bs delivery
The expressions of the proPO, LGBP and PE genes were significantly higher (p b 0.05) in both rVP28-bs and B. subtilis groups compared to the control (oral delivered by pBS coating feed pellets) (Fig. 2). However, no significant differences in proPO, LGBP and PE mRNA transcription levels of shrimp were observed between rVP28-bs and B. subtilis groups. 3.3. Specific hemocytic phagocytosis induced by rVP28-bs delivery Shrimp previously fed with rVP28-bs showed significantly more fluorescently-labelled WSSV in haemocytes (blood cells) than those fed with B. subtilis and pBS coating feed pellets (Fig. 3A). Similarly, the percentage of haemocytes phagocytosing WSSV was significantly higher (p b 0.001) in shrimp previously fed with rVP28-bs (48.2± 6.3) than those fed with B. subtilis (11.0 ± 3.5) and pBS (8.1 ± 2.5) coating
30
-3 ng
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B proPO mRNA -1 ¦Ìg total RNA ) (10
3.2. Effect of oral delivery on immune-related gene expressions
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0
PE mRNA -1 -3 (10 ng ¦Ìg total RNA )
In Experiment 1, oral delivery of rVP28-bs spores harboring the WSSV envelope protein VP28 resulted in a significantly lower cumulative mortality (p b 0.001) of L. vannamei at the challenge point of 7 days after 20-day feeding (Fig. 1). The calculated RPS value of rVP28-bs group was 83.3% compared with the positive control. Moreover, by oral delivery of B. subtilis spores, the number of survivors was also slightly increased after WSSV challenge leading to RPS value of 21.7% compared with the positive control. The negative control showed no mortality. Three randomly selected survivors from each group were tested for WSSV by PCR and tested negative.
35
rVP28-bs
B.subtilis
pBS
90 Fig. 2. Analysis of prophenoloxidase (proPO) (A), peroxinectin (PE) (B) and lipopolysaccharide and β-1,3-glucan binding protein (LGBP) (C) gene expression in hemocytes of the experimental groups by SYBR green real-time RT-PCR. Mean (± SEM) with different letters were significantly different (p b 0.05) among treatments.
80 70 60 50 40
feed pellets (Fig. 3B). There were no significant differences between B. subtilis and pBS groups for the phagocytic percentage of WSSV. Finally, there were no significant differences in all groups for the percentage phagocytosis of TSV (an unrelated pathogenic virus of shrimp) (Fig. 3).
30 20 10 0 0
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Fig. 1. Protective effects against WSSV of recombinant VP28 by oral delivery of spores of rVP28-bs strain to L. vannamei. Cumulative mortality rates of shrimp from the experimental groups were indicated. Shrimp were challenged 7 days after cessation of feeding coated pellets. The error bars indicate standard error of the mean (±SEM). Statistical significance was marked by the star.
3.4. Fate and dissemination of recombinant strain spores in L. vannamei In Experiment 2, shrimp were fed with 2.75 × 1010 spores in total, and measured the number of spores excreted. Our results showed that the majority of counts occurred within the first 24 h (4.0 × 1010), although spores were still detectable in the 6-day samples (7.2× 102). The cumulative number of spores excreted was 4.78 × 1010, and thus the ratio of spore counts to inoculum was 1.74. Moreover, we found that oral delivery produced relatively high levels of recombinant spore dissemination in the shrimp gut and stomach at 1, 3 and 7 days after
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A
Groups B. subtilis
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TSV
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Oral delivery rVP28-bs B.subtilis
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pBS
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Fig. 3. Evaluation of specificity of phagocytosis towards WSSV in L. vannamei hemocytes following feeding of rVP28-bs strain. Phagocytosing an unrelated virus (TSV) was also conducted as the control. (A) Hemocytes from rVP28-bs, B. subtilis and pBS groups phagocytosing FITC-labeled WSSV and TSV were examined under fluorescent microscope. (B) The percentage of haemocytes that showed phagocytic activity was recorded. The error bars indicate standard error of the mean (±SEM). Statistical significance was marked by the star.
feeding. The spores could also be detected at low levels in hepatopancreas at 3 days after feeding as well as in haemolymph at 3 and 7 days after feeding. It should be noted that spores cannot be detected at 14 days after feeding in all the above tissues (Fig. 4). Finally, no detectable spores appeared in the gill, heart and muscle of shrimp (data not shown). 4. Discussion In this study, challenge result showed that feeding rVP28-bs coated pellets conferred significantly higher protection of L. vannamei against WSSV (RPS: 83.3%) than that of the positive control, which was consistent with our previous results carried out in Procambarus clarkii (Fu et al., 2008) and F. chinensis (Fu et al., 2010). Furthermore, we have demonstrated that previous feeding of rVP28-bs spores to L. vannamei results in the increased phagocytic uptake of WSSV by the haemocytes. The phagocytosis of an unrelated virus of shrimp (TSV) by the same haemocytes was insignificantly changed compared to cells taken from shrimp fed with pBS coating feed. This suggests the stimulation of phagocytic activity, and thus leading to the high-level protection in shrimp after rVP28-bs ‘vaccination’ is selective or specific towards WSSV. Our results agree with those of another study using L. vannamei that observed enhanced phagocytosis of the bacterial shrimp pathogen, Vibrio harveyi, after challenge with the same species (Pope et al., 2011). To date, Dscam is one of the best candidates to explain the phenomenon of specific immune priming in invertebrate including shrimp (Chou et al., 2009, 2011; Dong et al., 2006; Watson et al., 2005). The mechanistic explanations for how the immune system accomplishes such heightened resistance to infection could arise the attention to the development of putative ‘vaccines’ aimed to boost growth and welfare in shrimp farming. Another observation of the current study is that humoral immunerelated genes (proPO, PE and LGBP) were significantly upregulated in both rVP28-bs and B. subtilis feeding groups compared to the control.
As an oral ‘vaccine’ carrier, B. subtilis might act as an adjuvant in the ‘vaccination’ leading to the enhanced humoral immune response in shrimp. However, the extremely high survival of L. vannamei challenged by WSSV could only be observed in the rVP28-bs group that also demonstrated a significantly high percentage of haemocytes phagocytosing WSSV. Taken together, it was indicated that the cellular response stimulated by rVP28-bs was the essential one to protect shrimp from virus infection, while the heightened humoral response only plays an assistant role in antiviral defense of shrimp. In Experiment 2, the total accumulated number of spores excreted over 144 h was higher than the original inoculum. Moreover, considerable numbers of spores were still being detected in the gut contents on day 6, and there was a gradual decline in spore counts over time. Our interpretation is that a proportion of the spores had germinated in the gut of shrimp, undergone one or more rounds of growth and replication, and then formed spores, which was similar to the results stated by Hoa et al. (2001) in a murine model. Thus, B. subtilis possibly exert their probiotic action (such as stimulation of humoral immune response) by a metabolic effect following spore germination. Another intriguing observation was that the B. subtilis spores harboring VP28 could be detected in the shrimp haemolymph after feeding. The dissemination of this live carrier in haemolymph by oral delivery might facilitate to present the antigen protein to the host defense system when secretedly express VP28. In conclusion, the heightened phagocytic activity, and thus the high-level survival of shrimp after rVP28-bs ‘vaccination’ are selective or specific towards WSSV. The phagocytosis enhanced by rVP28-bs was the essential one to protect shrimp from virus infection, while the heightened humoral response only plays an assistant role in antiviral defense of shrimp. Furthermore, as robust life forms, spores can survive transit across the gastrointestinal tract, germinate and disseminate in the haemolymph to present the antigen protein VP28 to host defense system before being excreted.
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Fig. 4. Spore dissemination examined by PCR detection (A) and immunofluorescence staining (B). Shrimp were sacrificed at 1, 3, 7 and 14 days after feeding and dissected for selected organs and tissues (gut, hepatopancreas, gill, stomach, heart, haemolymph and muscle) to detect in vivo dissemination. No detectable spores appeared in the gill, heart and muscle of shrimp (data not shown).
Acknowledgements This study was financially supported by the National Natural Science Foundation of China (31101921), the Zhejiang Provincial Natural Science Foundation of China (Y3090370 and R3110345), and the Science and Technology Development Plan of Hangzhou, China (20101032B47).
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