Stimulating the immune response of Litopenaeus vannamei using the phagocytosis activating protein (PAP) gene

High mortality in the shrimp farming industry is caused by several pathogens such as white spot syndrome virus (WSSV), yellow head virus (YHV) and Vibrio harveyi (V. harveyi). A PAP (Phagocytosis activating protein) gene able to activate phagocytosis of shrimp hemocytes was cloned into the eukaryotic expression vector phMGFP. In vitro expression was confirmed by transfection of PAP-phMGFP into CHO (Chinese Hamster Ovary) cells and the expression of the Green Fluorescent Protein (GFP) was observed. In order to activate the phagocytic activity of shrimp, 20, 40 and 80 μg/shrimp of this PAP-phMGFP vector were injected into Litopenaeus vannamei muscle. After challenged with WSSV, 40 μg/shrimp produced the highest relative percent survival (77.78 RPS). Analysis for the expression of the GFP gene in various tissues showed the expression mostly in the hemolymph of the immunized shrimp. The expression level of PAP and proPO (Prophenoloxidase) gene were highest at 7 days after immunization. This agreed with the efficiency of protection against WSSV that also occurred 7 days after immunization with the highest RPS of 86.61%. However there was no protection 30 days after immunization. Hemocytes of shrimp injected with PAP-phMGFP had 1.9 folds and 3 folds higher percentage phagocytosis and phagocytic index than the shrimp injected with PBS. Accordingly, copies of WSSV reduced in the PAP-phMGFP injected shrimp. In addition, PAP-phMGFP also protected shrimp against several pathogens: WSSV, YHV and V. harveyi, with RPS values of 86.61%, 63.34% and 50% respectively. This finding shows that the immune cellular defense mechanisms in shrimp against pathogens can be activated by injection of PAP-phMGFP and could indicate possible useful ways to begin to control this process.     

Dietary administration of a Gracilaria tenuistipitata extract enhances the immune response and resistance against Vibrio alginolyticus and white spot syndrome virus in the white shrimp Litopenaeus vannamei

The haemogram, phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, lysozyme activity, and the mitotic index of haematopoietic tissue (HPT) were examined after the white shrimp Litopenaeus vannamei had been fed diets containing the hot-water extract of Gracilaria tenuistipitata at 0 (control), 0.5, 1.0, and 2.0 g kg(-1) for 7-35 days. Results indicated that these parameters directly increased with the amount of extract and time, but slightly decreased after 35 days. RBs, SOD activity, and GPx activity reached the highest levels after 14 days, whereas PO and lysozyme activities reached the highest levels after 28 days. In a separate experiment, white shrimp L. vannamei, which had been fed diets containing the extract for 14 days, were challenged with Vibrio alginolyticus at 2 × 10(6) cfu shrimp(-1) and white spot syndrome virus (WSSV) at 1 × 10(3) copies shrimp(-1), and then placed in seawater. The survival rate of shrimp fed the extract-containing diets was significantly higher than that of shrimp fed the control diet at 72-144 h post-challenge. We concluded that dietary administration of the G. tenuistipitata extract at ≤1.0 g kg(-1) could enhance the innate immunity within 14 days as evidenced by the increases in immune parameters and mitotic index of HPT in shrimp and their enhanced resistance against V. alginolyticus and WSSV infections. Shrimp fed the extract-containing diets showed a higher and continuous increase in the humoral response indicating its persistent role in innate immunity.     

Protection of Penaeus monodon against white spot syndrome virus by oral vaccination

White spot syndrome virus (WSSV) occurs worldwide and causes high mortality and considerable economic damage to the shrimp farming industry. No adequate treatments against this virus are available. It is generally accepted that invertebrates such as shrimp do not have an adaptive immune response system such as that present in vertebrates. As it has been demonstrated that shrimp surviving a WSSV infection have higher survival rates upon subsequent rechallenge, we investigated the potential of oral vaccination of shrimp with subunit vaccines consisting of WSSV virion envelope proteins. Penaeus monodon shrimp were fed food pellets coated with inactivated bacteria overexpressing two WSSV envelope proteins, VP19 and VP28. Vaccination with VP28 showed a significant lower cumulative mortality compared to vaccination with bacteria expressing the empty vectors after challenge via immersion (relative survival, 61%), while vaccination with VP19 provided no protection. To determine the onset and duration of protection, challenges were subsequently performed 3, 7, and 21 days after vaccination. A significantly higher survival was observed both 3 and 7 days postvaccination (relative survival, 64% and 77%, respectively), but the protection was reduced 21 days after the vaccination (relative survival, 29%). This suggests that contrary to current assumptions that invertebrates do not have a true adaptive immune system, a specific immune response and protection can be induced in P. monodon. These experiments open up new ways to benefit the WSSV-hampered shrimp farming industry.     

Knocking down caspase-3 by RNAi reduces mortality in Pacific white shrimp Penaeus (Litopenaeus) vannamei challenged with a low dose of white-spot syndrome virus

Apoptosis has long been observed in viral target organs of white-spot syndrome virus (WSSV)-infected shrimp and whether the phenomenon helps the shrimp to survive the infection or is a factor leading to mortality is still controversial. If the shrimp mortality is a result of triggered apoptosis, then inactivation of caspase-3, a key protein in the induction of apoptosis, should improve shrimp survival upon challenge with WSSV. To test this prediction, we identified and characterized a caspase-3 homologue (cap-3) from the Pacific white shrimp Penaeus (Litopenaeus) vannamei and used this information to silence cap-3 expression by RNA interference prior to WSSV challenge. After confirming the efficacy of cap-3 silencing, its effects on mortality at high and low doses of WSSV were evaluated. In a high-dose WSSV challenge, cap-3 silencing had no significant effect on WSSV-induced mortality, except for a delay in mean time to death. However, at a low-dose WSSV challenge, cap-3 silencing correlated with a lower level of cumulative mortality, relative to silencing of a control gene, suggesting that apoptosis may exacerbate rather than decrease mortality in WSSV-challenged shrimp.     

Screening and selection of bacteria inhibiting white spot syndrome virus infection to Litopenaeus vannamei

A total of 173 bacterial strains were isolated from different sources at different regions such as fermented foods, shrimp guts, sea water, mangrove water, and sediments. These bacteria were screened against white spot syndrome virus (WSSV) infection in Palaemon paucidens. Based on mortality, white spot level, and healthiness, three bacterial strains were selected and identified using 16S rRNA gene sequencing. These bacterial strains were Bacillus subtilis KA1, B. licheniformis KA2, and B. subtilis KA3. WSSV challenge test in pilot scale was conducted using Litopenaeus vannamei with B. subtilis KA1 and B. subtilis KA3. The survival ratio of shrimp was 0% for WSSV control after 17th days, 84% for B. subtilis KA1 plus WSSV after 26th days, and 28% for B. subtilis KA3 with WSSV after 26th days. B. subtilis KA1 showed good growth at 18–37 °C in with and without 3% NaCl, and therefore can be applied to aquaculture at low to high temperatures. B. subtilis KA1 produced protease and lipase which can increase digestion to shrimp; exhibited antibacterial activity against Vibrio parahaemolyticus; and significantly increased the survival of WSSV challenged shrimps.     

Oral vaccination of baculovirus-expressed VP28 displays enhanced protection against White Spot Syndrome Virus in Penaeus monodon

White Spot Syndrome Virus (WSSV) is an infectious pathogen of shrimp and other crustaceans, and neither effective vaccines nor adequate treatments are currently available. WSSV is an enveloped dsDNA virus, and one of its major envelope proteins, VP28, plays a pivotal role in WSSV infection. In an attempt to develop a vaccine against WSSV, we inserted the VP28 gene into a baculovirus vector tailored to express VP28 on the baculovirus surface under the WSSV ie1 promoter (Bac-VP28). The Bac-VP28 incorporated abundant quantity (65.3 μg/ml) of VP28. Shrimp were treated by oral and immersion vaccination with either Bac-VP28 or wild-type baculovirus (Bac-wt). The treatment was followed by challenge with WSSV after 3 and 15 days. Bac-VP28 vaccinated shrimp showed significantly higher survival rates (oral: 81.7% and 76.7%; immersion: 75% and 68.4%) than Bac-wt or non-treated shrimp (100% mortality). To verify the protective effects of Bac-VP28, we examined in vivo expression of VP28 by immunohistochemistry and quantified the WSSV copy number by qPCR. In addition to that, we quantified the expression levels shrimp genes LGBP and STAT by real-time RT-PCR from the samples obtained from Bac-VP28 vaccinated shrimp at different duration of vaccine regime. Our findings indicate that oral vaccination of shrimp with Bac-VP28 is an attractive preventative measure against WSSV infection that can be used in the field.     

A novel protein with a fibrinogen-like domain involved in the innate immune response of Marsupenaeus japonicus

Fibrinogen-related proteins play important roles in innate immunity. We isolated a fibrinogen-related protein gene (MjFREP1) in kuruma shrimp Marsupenaeus japonicus. MjFREP1 encoded a protein of 270 amino acids, including a 223 amino acid fibrinogen-like domain. Quantitative real-time polymerase chain reaction analysis shows that MjFREP1 is mainly expressed in the gills and the expression is significantly upregulated by Vibrio anguillarum, Staphylococcus aureus, or white spot syndrome virus (WSSV) challenge. Recombinant MjFREP1 fibrinogen-like domain agglutinates Gram-positive bacteria Bacillus subtilis, Bacillus thuringiensis, Bacillus megaterium, and S. aureus in the presence of calcium ions. The fibrinogen-like domain of MjFREP1 binds peptidoglycans, LPS, bacteria, and the VP28 of WSSV. These results suggest that the MjFREP1 may play an important role in the shrimp immune response against different pathogens.     

以枯草芽孢杆菌递呈VP28对南美白对虾免疫相关基因表达和细胞特异性吞噬的影响

以枯草芽孢杆菌(Bacillus subtilis)为活载体口服递呈对虾白斑综合征病毒(WSSV)囊膜蛋白VP28, 评价其抗病毒感染能力、对南美白对虾免疫相关基因表达以及血淋巴细胞对病毒特异性吞噬的影响。经口服免疫枯草重组菌株B. subtilis-VP28攻毒后, 对虾的相对存活率达83.3%。为探讨重组菌株的抗病机理, 比较研究了免疫相关基因proPO(酚氧化酶原)、Peroxinectin(PE)和脂多糖--1, 3-葡聚糖结合蛋白(LGBP)基因的表达差异, 并进一步分析了血淋巴细胞吞噬活性和特异性。结果表明, B. subtilis-VP28菌液能显著提高(P 0.05)对虾proPO、PE和LGBP mRNA的表达水平和血细胞对WSSV的吞噬活性, B. subtilis组对免疫相关基因也有一定的激活作用, 而B. subtilis-VP28发酵上清液则能增加血细胞吞噬活性; 此外, B. subtilis-VP28菌液组血细胞对WSSV具有特异性吞噬作用。研究为枯草重组菌株B. subtilis-VP28抗WSSV感染作用及其作为特殊功能水产微生态制剂的应用提供了一定的科学依据。       

Enhanced survival of shrimp, Penaeus (Marsupenaeus) japonicus from white spot syndrome disease after oral administration of recombinant VP28 expressed in Brevibacillus brevis

White spot syndrome virus (WSSV) disease is a major threat to shrimp culture worldwide. Here, we assessed the efficacy of the oral administration of purified recombinant VP28, an envelope protein of WSSV, expressed in a Gram-positive bacterium, Brevibacillus brevis, in providing protection in shrimp, Penaeus japonicus, upon challenge with WSSV. Juvenile shrimp (2-3g in body weight) fed with pellets containing purified recombinant VP28 (50mug/shrimp) for 2weeks showed significantly higher survival rates than control groups when challenged with the virus at 3days after the last day of feeding. However, when shrimp were challenged 2weeks after the last day of feeding, survival rates decreased (33.4% and 24.93%, respectively). Survival rate was dose-dependent, increasing from 60.7 to 80.3% as the dose increased from 1 to 50mug/shrimp. At a dose of 50mug/shrimp, the recombinant protein provided protection as soon as 1day after feeding (72.5% survival). Similar results were obtained with larger-sized shrimp. These results show that recombinant VP28 expressed in a Gram-positive bacterium is a potential oral vaccine against WSSV.     

In vivo titration of white spot syndrome virus (WSSV) in specific pathogen-free Litopenaeus vannamei by intramuscular and oral routes

White spot syndrome virus (WSSV) is a devastating pathogen in shrimp aquaculture. Standardized challenge procedures using a known amount of infectious virus would assist in evaluating strategies to reduce its impact. In this study, the shrimp infectious dose 50% endpoint (SID50 ml(-1)) of a Thai isolate of WSSV was determined by intramuscular inoculation (i.m.) in 60 and 135 d old specific pathogen-free (SPF) Litopenaeus vannamei using indirect immunofluorescence (IIF) and 1-step polymerase chain reaction (PCR). Also, the lethal dose 50% endpoint (LD50 ml(-1)) was determined from the proportion of dead shrimp. The median virus infection titers in 60 and 135 d old juveniles were 10(6.8) and 10(6.5) SID50 ml(-1), respectively. These titers were not significantly different (p > or = 0.05). The titration of the WSSV stock by oral intubation in 80 d old juveniles resulted in approximately 10-fold reduction in virus titer compared to i.m. inoculation. This lower titer is probably the result of physical and chemical barriers in the digestive tract of shrimp that hinder WSSV infectivity. The titers determined by infection were identical to the titers determined by mortality in all experiments using both i.m. and oral routes at 120 h post inoculation (hpi), indicating that every infected shrimp died. The determination of WSSV titers for dilutions administered by i.m. and oral routes constitutes the first step towards the standardization of challenge procedures to evaluate strategies to reduce WSSV infection.     

Increased susceptibility of white spot syndrome virus-infected Litopenaeus vannamei to Vibrio campbellii

The concept of polymicrobial disease is well accepted in human and veterinary medicine but has received very little attention in the field of aquaculture. This study was conducted to investigate the synergistic effect of white spot syndrome virus (WSSV) and Vibrio campbellii on development of disease in specific pathogen-free (SPF) shrimp Litopenaeus vannamei. The juvenile shrimp were first injected with WSSV at a dose of 30 SID(50) shrimp(-1) (SID(50) = shrimp infectious dose with 50% endpoint) and 24 h later with 10(6) colony-forming units (cfu) of V. campbellii shrimp(-1). Controls receiving just one of the pathogens or negative inocula were included. In the treatment with WSSV only, shrimp started to die at 48-108 h post injection (hpi) and cumulative mortality reached 100% at 268-336 hpi. In the treatment with only V. campbellii injection (10(6) cfu shrimp(-1)), cumulative mortality reached 16.7%. Shrimp in the dual treatment died very quickly after V. campbellii injection and 100% cumulative mortality was obtained at 72-96 hpi. When WSSV-injected shrimp were given sonicated V. campbellii instead of live V. campbellii, no synergistic effect was observed. Density of V. campbellii in the haemolymph of co-infected moribund shrimp collected 10 h after V. campbellii injection was significantly higher than in shrimp injected with V. campbellii only (P < 0.01). However, there was no difference in WSSV replication between shrimp inoculated with WSSV only compared with dually inoculated ones. This study revealed that prior infection with WSSV enhances the multiplication and disease inducing capacity of V. campbellii in shrimp.     

Silencing VP28 Gene of White Spot Syndrome Virus of Shrimp by Bacterially Expressed dsRNA

An in vivo expression system to produce large amounts of virus-derived dsRNAs in bacteria to provide a practical control of white spot syndrome virus (WSSV) in shrimp was developed. The bacterially synthesized dsRNA specific to VP28 gene of WSSV promoted gene-specific interference with the WSSV infection in shrimp. Virus infectivity was significantly reduced in WSSV-challenged shrimp injected with VP28-dsRNA and 100% survival was recorded. The inhibition of the expression of WSSV VP28 gene in experimentally challenged animals by VP28-dsRNA was confirmed by RT-PCR and Western blot analyses. Furthermore, we have demonstrated the efficacy of bacterially expressed VP28-dsRNA to silence VP28 gene expression in SISK cell line transfected with eukaryotic expression vector (pcDNA3.1) inserted with VP28 gene of WSSV. The expression level of VP28 gene in SISK cells was determined by fluorescent microscopy and ELISA. The results showed that the expression was significantly reduced in cells transfected with VP28dsRNA, whereas the cells transected with pcDNA-VP28 alone showed higher expression. The in vivo production of dsRNA using prokaryotic expression system could be an alternative to in vitro method for large-scale production of dsRNA corresponding to VP28 gene of WSSV for practical application to control the WSSV in shrimp farming.     

Immunological responses of Penaeus monodon to DNA vaccine and its efficacy to protect shrimp against white spot syndrome virus (WSSV)

White spot disease is an important viral disease caused by white spot syndrome virus (WSSV) and is responsible for huge economic losses in the shrimp culture industry worldwide. The VP28 gene encoding the most dominant envelope protein of WSSV was used to construct a DNA vaccine. The VP28 gene was cloned in the eukaryotic expression vector pcDNA3.1 and the construct was named as pVP28. The protective efficiency of pVP28 against WSSV was evaluated in Penaeus monodon by intramuscular challenge. In vitro expression of VP28 gene was confirmed in sea bass kidney cell line (SISK) by fluorescence microscopy before administering to shrimp. The distribution of injected pVP28 in different tissues of shrimp was studied and the results revealed the presence of pVP28 in gill, head soft tissue, abdominal muscle, hemolymph, pleopods, hepatopancreas and gut. RT-PCR and fluorescence microscopy analyses showed the expression of pVP28 in all these tissues examined. The results of vaccination trials showed a significantly higher survival rate in shrimp vaccinated with pVP28 (56.6-90%) when compared to control groups (100% mortality). The immunological parameters analyzed in the vaccinated and control groups revealed that the vaccinated shrimp showed significantly high level of prophenoloxidase and superoxide dismutase (SOD) when compared to the control groups. The high levels of prophenoloxidase and superoxide dismutase (SOD) might be responsible for developing resistance against WSSV in DNA vaccinated shrimp.     

Polychaete worms--a vector for white spot syndrome virus (WSSV)

The present work provides the first evidence of polychaete worms as passive vectors of white spot syndrome virus (WSSV) in the transmission of white spot disease to Penaeus monodon broodstocks. The study was based on live polychaete worms, Marphysa spp., obtained from worm suppliers/worm fishers as well as samples collected from 8 stations on the northern coast of Tamilnadu (India). Tiger shrimp Penaeus monodon broodstock with undeveloped ovaries were experimentally infected with WSSV by feeding with polychaete worms exposed to WSSV. Fifty percent of polychaete worms obtained from worm suppliers were found to be WSSV positive by 2-step PCR, indicating high prevalence of WSSV in the live polychaetes used as broodstock feed by hatcheries in this area. Of 8 stations surveyed, 5 had WSSV positive worms with prevalence ranging from 16.7 to 75%. Polychaetes collected from areas near shrimp farms showed a higher level of contamination. Laboratory challenge experiments confirmed the field observations, and > 60% of worms exposed to WSSV inoculum were proved to be WSSV positive after a 7 d exposure. It was also confirmed that P. monodon broodstock could be infected with WSSV by feeding on WSSV contaminated polychaete worms. Though the present study indicates only a low level infectivity in wild polychaetes, laboratory experiments clearly indicated the possibility of WSSV transfer from the live feed to shrimp broodstock, suggesting that polychaete worms could play a role in the epizootiology of WSSV.     

Viral interference between infectious hypodermal and hematopoietic necrosis virus and white spot syndrome virus in Litopenaeus vannamei

White spot syndrome virus (WSSV) is highly virulent and has caused significant production losses to the shrimp culture industry over the last decade. Infectious hypodermal and hematopoietic necrosis virus (IHHNV) also infects penaeid shrimp and, while being less important than WSSV, remains a major cause of significant production losses in Litopenaeus vannamei (also called Penaeus vannamei) and L. stylirostris (also called Penaeus stylirostris). These 2 viruses and their interactions were previously investigated in L. stylirostris. We report here laboratory challenge studies carried out to determine if viral interference between IHHNV and WSSV also occurs in L. vannamei, and it was found that experimental infection with IHHNV induced a significant delay in mortality following WSSV challenge. L. vannamei infected per os with IHHNV were challenged with WSSV at 0, 10, 20, 30, 40 and 50 d post-infection. Groups of na?ve shrimp infected with WSSV alone died in 3 d whereas shrimp pre-infected with IHHNV for 30, 40 or 50 d died in 5 d. Real-time PCR analysis showed that the delay correlated to the IHHNV load and that WSSV challenge induced a decrease in IHHNV load, indicating some form of competition between the 2 viruses.