Studies on the rotifer (Brachionus urceus Linnaeus, 1758) as a vector in white spot syndrome virus (WSSV) transmission

The rotifer Brachionus urceus (Linnaeus, 1758) was experimentally infected with the white spot syndrome virus (WSSV) by the virus-phytoplankton adhesion route in order to assess the possibility of rotifer acting as a vector of WSSV to infect the shrimp Fenneropenaeus chinensis (Osbeck, 1765) larvae at zoea stage III. The nested-PCR test revealed WSSV-positive results in the rotifers exposed to WSSV by the virus-phytoplankton adhesion route. Among 10 replicates in the infection treatment, 40% of F. chinensis larvae became WSSV-positive when fed with WSSV-positive rotifers, whereas all were WSSV-negative for F. chinensis when fed with WSSV-free rotifers. Though the mortality of shrimp larvae in the infection treatment (39.47 ± 15.44%) was higher than that in the control treatment (34.67 ± 15.11%), there was no significant difference in the mortality between them (P > 0.05). These results indicated that the rotifer could serve as a vector in WSSV transmission when ingested.     

基于GFM和GAMM模型分析对虾白斑综合征(WSSV)对黄海和东海北部水域虾类生物量的影响

虾类是海洋生态系统功能群的重要组成部分,其生物量变化受到多重因素的影响。本研究在开展黄海和东海北部水域虾类白斑综合征病毒(white spot syndrome virus, WSSV)流行病学调查的基础上,利用梯度随机森林模型(gradient random forest model, GFM)和广义加性混合模型(generalized additive mixed models, GAMM),分析了2016—2018年间黄海和东海北部水域WSSV流行对虾类生物量的影响。分子检测结果显示,调查所获取的26种虾类中,11种被检测为WSSV阳性;2016、2017和2018年WSSV阳性采样站点的比率分别为48.40%、38.75%和21.74%,虾类样品中WSSV阳性检出比率分别为16.86%、9.60%和4.80%。GFM模型分析显示,解释变量“阳性样品数的对数(ln_posi)”对响应变量“虾类生物量的对数(ln_Abu)”的重要性最高。GAMM分析中,根据赤池信息准则(Akaike information criterion, AIC)最小原则筛选出的最优模型为：ln_Abu~WSSV阳性率(P_rate)+ln_posi+经度(Long),该模型中ln_posi和P_rate是影响虾类生物量的极显著相关因子,ln_Abu随着P_rate的升高而降低。研究表明,WSSV在黄海和东海北部水域虾类中流行,推测对该海域的虾类生物量存在影响。     

Silencing Pacific white shrimp Litopenaeus vannamei LvRab7 reduces mortality in brooders challenged with white spot syndrome virus

White spot syndrome virus (WSSV) is a major threat for farmed shrimp worldwide. RNA interference (RNAi) is the most recent tool against viral diseases. Rab7 silencing effectively inhibited virus infections in juvenile shrimp, but the antiviral effect in brooders remains unknown. This study found a homologue Penaeus monodon Rab7 gene in Litopenaeus vannamei brooders from Mexico. Sequence identity was >99% to a Thai LvRab7 sequence and >94% to Rab7 sequences from P. monodon or Marsupenaeus japonicus. Animals treated with a partial (494 bp) or a complete (618 bp) LvRab7 dsRNA sequences and challenged 48 h post treatment (hpt) with a high WSSV dose showed 80–88% mortality respectively. Shrimp treated with 4 or 20 μg LvRab7 dsRNA and challenged with a WSSV high dose had 80% mortality each, but it was reduced to 33% and 40%, respectively, with a low dose. Efficacy of dsRNA to reduce shrimp mortality was dependent on virus dose used regardless of dsRNA concentration. A significant reduction in LvRab7 mRNA levels was observed at 120 hpt. In conclusion, silencing LvRab7 in brooders showed a mild antiviral effect against a WSSV challenge at 48 hpt.     

Effects of acute change in salinity and moulting on the infection of white leg shrimp (Penaeus vannamei) with white spot syndrome virus upon immersion challenge

In the field, moulting and salinity drop in the water due to excessive rainfall have been mentioned to be risk factors for WSSV outbreaks. Therefore, in this study, the effect of an acute change in environmental salinity and shedding of the old cuticle shell on the susceptibility of Penaeus vannamei to WSSV was evaluated by immersion challenge. For testing the effect of abrupt salinity stress, early premoult shrimp that were acclimated to 35 g L^?1 were subjected to salinities of 50 g L^?1, 35 g L^?1, 20 g L^?1, 10 g L^?1 and 7 g L^?1 or 5 g L^?1 and simultaneously exposed to 10^5.5 SID₅₀ mL^?1 of WSSV for 5 h, after which the salinity was brought back to 35 g L^?1. Shrimp that were transferred from 35 g L^?1 to 50 g L^?1, 35 g L^?1 and 20 g L^?1 did not become infected with WSSV. Shrimp became infected with WSSV after an acute salinity drop from 35 g L^?1 to 10 g L^?1 and lower. The mortality in shrimp, subjected to a salinity change to 10 g L^?1, 7 g L^?1 and 5 g L^?1, was 6.7%, 46.7% and 53.3%, respectively (P < 0.05)_. For testing the effect of moulting, shrimp in early premoult, moulting and post‐moult were immersed in sea water containing 10^5.5 SID₅₀ mL^?1 of WSSV. The resulting mortality due to WSSV infection in shrimp inoculated during early premoult (0%), ecdysis (53.3%) and post‐moult (26.72%) demonstrated that a significant difference exists in susceptibility of shrimp during the short moulting process (P < 0.05). The findings of this study indicate that during a drop in environmental salinity lower than 10 g L^?1 and ecdysis, shrimp are at risk for a WSSV infection. These findings have important implications for WSSV control measures.     

Passive immunity in tiger shrimp (Penaeus monodon) fed with monoclonal antibody to white spot syndrome virus

A study was conducted on the stability of monoclonal antibody (MAb) in the hepatopancreas and hemolymph of Penaeus monodon and its effect on protection against white spot syndrome virus (WSSV) upon challenge. MAb C-5 raised against WSSV was purified and coated onto a commercial shrimp feed at dosages of 5, 10 and 15 mg/kg feed. The feed was fed to P. monodon and stability of the MAb in hepatopancreas and hemolymph was determined by immunodot and Western blot. Immunodot results indicated the presence of MAb for 2 h post-feeding in hepatopancreas and hemolymph which was dose-dependent. MAb was also detected in hemolymph by Western blot up to 1 h post-feeding. Shrimp fed with MAb were challenged with WSSV by oral and injection methods. In shrimp fed with 15 mg antibody/kg feed (0.45 μg MAb/g shrimp/day) WSSV infection significantly delayed both in oral and injection challenges with a survival of 65 and 70 % (p < 0.05), respectively, during 15 days post-challenge. MAb was stable in shrimp for passive immunization against WSSV and could be a potential tool for prophylaxis against the virus.     

White spot syndrome virus (WSSV) infection and immunity responses in white shrimp (Litopenaeus vannamei) exposed to sublethal levels of metals

To determine if exposure to a sublethal mixture of metals (Cd, Cu, Fe, Mn, Pb and Zn) increases susceptibility to White spot syndrome virus (WSSV) infection, Litopenaeus vannamei juveniles were fed WSSV‐infected shrimp tissues after 21 days of exposure to the metal mixture (WS‐MM treatment). Other treatments consisted of shrimp not exposed to metals and fed infected tissues (WS), and shrimp fed healthy tissues and exposed (MM) or not exposed to metals (C). The presence of viral DNA and inclusion bodies was detected at 32 hr postinfection (hpi) in the stomach epithelium of shrimp from the WS treatment, and eight hours later in shrimp from the WS‐MM treatment, possibly because of an initial negative effect of metals in viral replication. At 40 hpi, the severity of infection represented by the histopathological index increased in both WS and WS‐MM treatments, and values were higher in WS‐MM than in WS shrimp at the end of the experiment. From 56 hpi to the end of experiment, total hemocyte counts were lower in both WS‐MM and WS treatments, and concentrations were particularly low in WS‐MM shrimp. Conversely, phenoloxidase activity was higher in the WS‐MM treatment from 32 to 56 hpi, suggesting a possible role of the prophenoloxidase (proPO) system in the antiviral defense against WSSV. The presence of heavy metals at sublethal concentrations may increase shrimp susceptibility to WSSV; this is supported by a decrease in circulating hemocytes, an increase in the humoral response, and the development of a higher number of WSSV inclusion bodies.     

Effects of total ammonia,temperature and salinity on the mortality and viral replication of WSSV‐infected Chinese shrimp (Fenneropenaeus chinensis)

In this study, we evaluated the effects of three factors, total ammonia, temperature and salinity, on the mortality of and viral proliferation in white spot syndrome virus (WSSV)‐infected Chinese shrimp. Shrimp maintained in 30‰ seawater at 25°C with 0.34 mg L^?1 total ammonia (control group) were injected with approximately 20 WSSV particles per shrimp and subsequently subjected to the following conditions: 30‰ seawater at 25°C, with 6 mg L^?1 (N1 group) or 14 mg L^?1 (N2 group) total ammonia; 30‰ seawater at 18°C (T1 group) or 30°C (T2 group), with 0.34 mg L^?1 total ammonia and 20‰ (S1 group) or 40‰ (S2 group) seawater at 25°C, with 0.34 mg L^?1 total ammonia. An anova analysis revealed that the cumulative mortality of WSSV‐infected Chinese shrimp was significantly lower when reared in the T1 group compared to that of the T2 and control group. Similarly, the mortality of the shrimp in the S1 group was also significantly lower than that of the S2 and control group. No significant differences were detected among the N1, N2 and control groups. Accordingly, the WSSV level in the T1 and S1 groups was significantly lower than those in the control, T2 or S2 groups respectively. No significant differences in viral loads were detected among the control, N1 and N2 groups. The transfer of Chinese shrimp to lower temperature and lower salinity enhanced their resistance to WSSV infection, whereas a change in the concentration of total ammonia had no significant effect on the mortalities and viral loads of WSSV‐infected shrimp.     

Pre-exposure to infectious hypodermal and haematopoietic necrosis virus or to inactivated white spot syndrome virus (WSSV) confers protection against WSSV in Penaeus vannamei (Boone) post-larvae

Larvae and post-larvae of Penaeus vannamei (Boone) were submitted to primary challenge with infectious hypodermal and haematopoietic necrosis virus (IHHNV) or formalin-inactivated white spot syndrome virus (WSSV). Survival rate and viral load were evaluated after secondary per os challenge with WSSV at post-larval stage 45 (PL45). Only shrimp treated with inactivated WSSV at PL35 or with IHHNV infection at nauplius 5, zoea 1 and PL22 were alive (4.7% and 4%, respectively) at 10 days post-infection (p.i.). Moreover, at 9 days p.i. there was 100% mortality in all remaining treatments, while there was 94% mortality in shrimp treated with inactivated WSSV at PL35 and 95% mortality in shrimp previously treated with IHHNV at N5, Z1 and PL22. Based on viral genome copy quantification by real-time PCR, surviving shrimp previously challenged with IHHNV at PL22 contained the lowest load of WSSV (0-1x10(3) copies microg-1 of DNA). In addition, surviving shrimp previously exposed to inactivated WSSV at PL35 also contained few WSSV (0-2x10(3) copies microg-1 of DNA). Consequently, pre-exposure to either IHHNV or inactivated WSSV resulted in slower WSSV replication and delayed mortality. This evidence suggests a protective role of IHHNV as an interfering virus, while protection obtained by inactivated WSSV might result from non-specific antiviral immune response.     

TaqMan real-time PCR assay for relative quantification of white spot syndrome virus infection in Penaeus monodon Fabricius exposed to ammonia

White spot disease is caused by a highly virulent pathogen, the white spot syndrome virus (WSSV). The disease is usually triggered by changes in environmental parameters causing severe losses to the shrimp industry. This study was undertaken to quantify the relative WSSV load in shrimp exposed to ammonia, using a TaqMan‐based real‐time PCR, and their subsequent susceptibility to WSSV. Shrimp were exposed to different levels of total ammonia nitrogen (TAN) (8.1, 3.8 and 1.1 mg L^?1) for 10 days and challenged with WSSV by feeding WSSV‐positive shrimp. WSSV was detected simultaneously in haemolymph, gills and pereopods at four hours post‐infection. The TaqMan real‐time PCR assay showed a highly dynamic detection limit that spanned over 6 log₁₀ concentrations of DNA and high reproducibility (standard deviation 0.33–1.42) and small correlation of variability (CV) (1.89–3.85%). Shrimp exposed to ammonia had significantly higher (P < 0.01) WSSV load compared to the positive control, which was not exposed to ammonia. Shrimp exposed to 8.1 mg L^?1 of TAN had the highest (P < 0.01) WSSV load in all three organs in comparison with those exposed to 3.8 and 1.1 mg L^?1 of TAN. However, haemolymph had significantly higher (P < 0.01) viral load compared to the gills and pereopods. Results showed that shrimp exposed to ammonia levels as low as 1.1 mg L^?1 (TAN) had increased susceptibility to WSSV.     

Transmission of white spot syndrome virus (WSSV) from Dendronereis spp. (Peters) (Nereididae) to penaeid shrimp

Dendronereis spp. (Peters) (Nereididae) is a common polychaete in shrimp ponds built on intertidal land and is natural food for shrimp in traditionally managed ponds in Indonesia. White spot syndrome virus (WSSV), an important viral pathogen of the shrimp, can replicate in this polychaete (Desrina et al. 2013); therefore, it is a potential propagative vector for virus transmission. The major aim of this study was to determine whether WSSV can be transmitted from naturally infected Dendronereis spp. to specific pathogen‐free (SPF) Pacific white shrimp Litopenaeus vannamei (Boone) through feeding. WSSV was detected in naturally infected Dendronereis spp. and Penaeus monodon Fabricius from a traditional shrimp pond, and the positive animals were used in the current experiment. WSSV‐infected Dendronereis spp. and P. monodon in a pond had a point prevalence of 90% and 80%, respectively, as measured by PCR. WSSV was detected in the head, gills, blood and mid‐body of Dendronereis spp. WSSV from naturally infected Dendronereis spp was transmitted to SPF L. vannamei and subsequently from this shrimp to new naïve‐SPF L. vannamei to cause transient infection. Our findings support the contention that Dendronereis spp, upon feeding, can be a source of WSSV infection of shrimp in ponds.     

The incidence of suspected white spot syndrome virus in semi‐intensive and extensive shrimp farms in Bangladesh: implications for management

The study was conducted to assess key factors influencing suspected white spot syndrome virus (WSSV) disease and associated shrimp production and economic performance in three contrasting black tiger shrimp (Penaeus monodon) culture technologies promoted by the United States Agency for International Development funded Shrimp Quality Support Project (SQSP) in Bangladesh. A total of 350 traditional, 315 Modified Traditional Technology1 (MTT1), 36 MTT2 and 88 Closed System Technology (CST) farmers from 10 sub‐districts in three districts of Khulna division were surveyed following random sampling at the end of the project. Binomial probit regression analysis revealed that smaller newly constructed ponds (known locally as gher) were less susceptible to WSSV, provided aquatic weeds were controlled using chemicals. Removal of sludge from ghers also had a positive effect, irrespective of technology and location. It was also shown that stocking of screened shrimp postlarvae (PL) does not guarantee protection against WSSV (t = 1.39, P > 0.05). Higher shrimp production was obtained by farmers practicing CST, followed by those operating MTTs and traditional technology respectively. Farmers who adopted CST also gained higher profitability followed by those operating MTT1, MTT2 and traditional technology.     

Anti-viral activity of methyl 1-chloro-7-methyl-2-propyl-1h-benzo[d] imidazole-5-carboxylate against white spot syndrome virus in freshwater crab (Paratelphusa hydrodromous)

Shrimp farming industries are subjected to severe economic loss due to a disease called white spot syndrome, a viral disease caused by white spot syndrome virus (WSSV) in penaeid shrimp. Numerous active compounds in the market possess anti-viral activity against the white spot syndrome virus, yet the issue remains unsolved. The present study was carried out to determine the anti-viral activity of methyl 1-chloro-7-methyl-2-propyl-1h-benzo[d] imidazole-5-carboxylate (C₁₃H₁₅N₂O₂Cl) against WSSV. The anti-viral activity of the synthetic compound was determined in freshwater crabs. Crabs were divided into three different experimental groups: healthy control groups (N.C.) received NTE buffer, positive control group (P.C.) crabs received WSSV, and treatment group crabs received WSSV along with synthetic weight compound. Experimental groups were observed for 30 days post-infection. Three different organs (gills, muscles, and head soft tissue (HST)) were dissected from all three groups and analyzed using molecular-based techniques, including polymerase chain reaction (PCR), Western blot, and histopathology. Clinical signs of WSSV were observed in the positive and N.C. groups; however, the treatment group showed a 100% survival rate. Confirmation was done using PCR, Western blot, and histopathology. These results demonstrated that the given synthetic compound has significant anti-viral activity against WSSV.

     

Development of a monoclonal antibody‐based flow‐through immunoassay (FTA) for detection of white spot syndrome virus (WSSV) in black tiger shrimp Penaeus monodon

A flow‐through immunoassay (FTA), an improved version of immunodot, was developed using a nitrocellulose membrane baked onto adsorbent pads enclosed in a plastic cassette to detect white spot syndrome virus (WSSV) in shrimp. Sharp purple dots developed with WSSV against the white background of the nitrocellulose membrane. The detection limits of WSSV by the FTA and immunodot were 0.312 and 1.2 μg mL^?1 crude WSSV protein, respectively. The FTA could be completed in 8–10 min compared with 90 min for immunodot. The FTA was 100 times more sensitive than 1‐step polymerase chain reaction (PCR) and in between that of the 1‐ and 2‐step PCR protocol recommended by the Office of International Epizootics (OIE). In experimental, orally infected shrimp post‐larvae, WSSV was first detected 14, 16 and 18 h post‐infection (hpi) by FTA, immunodot and one‐step PCR, respectively. The FTA detected WSSV 2 and 4 h earlier than immunodot and one‐step PCR, respectively. The FTA was more sensitive (25/27) than one‐step PCR (23/27) and immunodot (23/27) for the detection of WSSV from white spot disease outbreak ponds. The reagent components of the FTA were stable giving expected results for 6 m at 4–8 °C. The FTA is available as a rapid test kit called ‘RapiDot’ for the early detection of WSSV under field conditions.     

A synthetic antibacterial peptide from Mytilus galloprovincialis reduces mortality due to white spot syndrome virus in palaemonid shrimp

White spot syndrome virus (WSSV) isolated from Penaeus monodon was found to be highly infective for the western Mediterranean shrimp, Palaemon sp. Using polymerase chain reaction (PCR), it was demonstrated that such shrimp are not naturally carriers of WSSV. Following challenge with virus, mortality reached 100% 3.5-4 days after injection at 22 degrees C. Incubation of infected shrimp at 10 degrees C totally suppressed the mortality which rapidly developed when shrimp were returned to 18 or 22 degrees C. Preincubation of WSSV with mature synthetic mytilin significantly reduced shrimp mortality with a 50% efficient dose of about 5 microM. Survival of shrimp was not due to the development of an active mechanism of defence as re-injection of WSSV produced the same mortality pattern. Mortality was probably due to WSSV replication as dot blot failed to detect viral DNA in the injection sample but was positive 1 day post-injection. Protection by mytilin was by interaction at the virus level, preventing replication as no WSSV nucleic acid was detected by PCR even after 7 days in shrimp injected with WSSV preincubated with 10 or 50 microM mytilin.     

Genetic variation of white spot syndrome virus (WSSV) in Pacific white shrimp Litopenaeus vannamei (Boone 1931) culture of Thailand

White spot syndrome virus (WSSV) is highly pathogenic to penaeid shrimp and has caused significant economic losses in the shrimp farming industry in Thailand. Genotyping analysis was done in 124 WSSV isolates from cultured Pacific white shrimp Litopenaeus vannamei. These samples were obtained during 2007–2014 from eight provinces in Thailand. We investigated five variable loci in the virus genome: deletions in two variable regions, VR14/15 and VR23/24, and three variable number tandem repeats (VNTR) located in open reading frame (ORF) 75, 125 and 94. WSSV genotype was characterized as (X_14/15, X_23/24) (N₇₅‐N₁₂₅‐N₉₄) where X is the number of base pair deletion in the variable region and N is the number of repeat units (RUs) in a specific ORF. The deletion pattern in VR14/15 and VR23/24 regions characterized three WSSV genotypes. The most prevalent genotype was (5950_14/15, 10971_23/24), and it was found in all studied areas. At least 33 genotypes of WSSV were analysed based on 3 VNTR loci, indicating that the VNTRs of WSSV genome are highly variable. From 124 WSSV samples, two samples presented the characteristic of all five variable loci similar to WSSV collected during 2010 in Saudi Arabia (5950_14/15, 10971_23/24) (3₇₅‐6₁₂₅‐7₉₄). Many different WSSV genotypes shown in this study as compared to previously reported genotypes in Thailand suggests current status of disease epidemiology, as well as probable movements of WSSV between countries.     

Prevalence of white spot syndrome virus infection detected by one-step and nested PCR in selected tiger shrimp (<Emphasis Type="Italic">Penaeus monodon</Emphasis>) hatcheries

White spot syndrome (WSS) is considered as a great threat to commercial farming of the tiger shrimp (Penaeus monodon). The causal agent of WSS is a DNA virus called white spot syndrome virus (WSSV). The prevalence of this dreadful virus infection has been studied in five randomly selected hatcheries located in the Cox’s Bazar district of Bangladesh. Both one-step and nested polymerase chain reaction (PCR) involving two pairs of primers, namely, 146F1/146R1 and 146F2/146R2, amplifying the 1447 bp and 941 bp fragments, respectively, were conducted to detect the WSSV. Out of 60 randomly collected shrimps, 12 (20%) were found to be positive by one-step PCR, while 18 (30%) were found to be positive by nested PCR. The nested PCR was found to be much more sensitive than the one-step PCR. The shrimp specimens showing clinical signs of WSS were positive for WSSV by both one-step and nested PCR. Some of the apparently healthy samples were also found to be positive for WSSV by nested PCR. Among the two primer-pairs, the inner pair amplifying the 941 bp fragment was more sensitive than the outer primer pair amplifying the 1447 bp fragment when used in one-step PCR.     

Probiotic microorganisms and antiviral plants reduce mortality and prevalence of WSSV in shrimp (Litopenaeus vannamei) cultured under laboratory conditions

The protective effect of a probiotic mixture (PM) and antiviral plants, against the white spot syndrome virus (WSSV) in Litopenaeus vannamei , was evaluated in three experiments. The PM was composed of four lactic acid bacteria (LAB) and one yeast strain. The plant mixture was composed of Ocimum sanctum and commercial antiviral plants (VPH^®, HSV^®). Shrimp in each experiment (weighing 2.7±0.7, 11.5±1.3, 11.70±2.5 g) were cultured in 120-L plastic tanks and fed twice a day with commercial feed plus additives (plants or bacteria and yeast). Animals were monitored for the occurrence of WSSV by single-step and nested PCR. The PM and powdered antiviral plants added to the commercial feed showed an increase in survival and a decrease in the prevalence of WSSV in shrimp. The results showed that both the PM and the powdered antiviral plants can provide protection for shrimp against WSSV.     

Assessment of transmission risk in WSSV-infected shrimp Litopenaeus vannamei upon cooking

White spot syndrome virus has been a threat to the global shrimp industry since it was discovered in Taiwan in 1992. Thus, shrimp-producing countries have launched regulations to prevent import of WSSV-infected commodity shrimp from endemic areas. Recently, cooked shrimp that is infected with WSSV tested positive by PCR. However, there is no study to determine the infectivity of WSSV in cooked shrimp that tested positive by PCR. In the present study, WSSV-infected shrimp were cooked at boiling temperature for different times including 0, 1, 3, 5, 10 and 30 min. Upon exposure to boiling temperature, WSSV-infected shrimp were fed to SPF shrimp (Litopenaeus vannamei). The result showed experimentally challenged shrimp from 0-min treatment (positive control) indeed got infected with WSSV. However, experimentally challenged shrimp that were fed tissues boiled at 1, 3, 5, 10 and 30 min were not infected with WSSV. Mortality data showed that only the positive control (0-min) treatment displayed high mortality, whereas no mortality was observed in any other treatment category. These findings suggest that cooking shrimp at boiling temperature for at least 1 min might prevent any potential spread of WSSV from endemic countries to other geographical areas where WSSV has not yet been reported.