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Simultaneous detection of Macrobrachium rosenbergii nodavirus and extra small virus by a single tube, one-step multiplex RT-PCR assay
Abstract
Post-larvae of Macrobrachium rosenbergii infected with white tail disease were collected from hatcheries and nursery ponds in India. The causative organisms have been identified as Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV). A one-step multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) has been developed to detect these viruses simultaneously in naturally and experimentally infected prawns. Several parameters were assayed in order to optimize the protocol for simultaneous detection. Naturally and experimentally infected prawns showed two prominent bands of 681 and 500 bp for MrNV and XSV, respectively, as in separate RT-PCR assays. Experimentally infected adult prawns showed two bands for these two viruses in all the organs, except hepatopancreas and eyestalk, as seen in normal RT-PCR. The sensitivity test carried out on the primer sets of MrNV and XSV revealed that these primers could simultaneously detect the two viruses at a level of 25 fg of total RNA prepared from infected samples using this multiplex RT-PCR protocol.
... On the other hand, recent work suggests that MrNV is the major cause of pathology in the mixed infection (Zhang et al., 2006). Detection methods for these viruses using RT-PCR, multiplex RT-PCR, Taqman realtime RT-PCR, in situ hybridization, dot blot hybridization, double antibody sandwich enzyme-linked immunosorbent assay, loop-mediated isothermal amplification, and high resolution melt duplex RT-PCR have been established (Pillai et al., 2006;Puthawibool et al., 2010;Romestand and Bonami, 2003;Senapin et al., 2010;Sri Widada et al., 2003;Yoganandhan et al., 2005;Zhang et al., 2006). Aquaculture 338-341 (2012) (Sudhakarana et al., 2006), but M. rosenbergii was the only natural host reported until the recent description of WTD outbreaks with high mortality in postlarvae of P. monodon and P. indicus in India (Ravi et al., 2009). ...
... RT-PCR detection of MrNV and XSV was carried out separately using single-step RT-PCR methods previously described (Yoganandhan et al., 2005). These methods targeted capsid coding genes of the respective viral genome sequences. ...
... All of the samples we processed were negative for both IMNV and PvNV (Senapin et al., 2011). After reports of MrNV infections in penaeid shrimp (Ravi et al., 2009;Sudhakarana et al., 2006), we re-examined our archived RNA extracts for MrNV using the nested RT-PCR detection method described in Materials and methods with the first round RT-PCR reaction based on an established protocol (Yoganandhan et al., 2005). A total of 36 P. vannamei samples collected from ponds exhibiting white muscles and high mortality from five Asian countries were examined (Table 1). ...
Macrobrachium rosenbergii nodavirus (MrNV) is well-known as a major pathogen that causes whitened muscles and high mortality in the freshwater prawn Macrobrachium rosenbergii. Recently, it has also been reported to cause white muscles and high mortality in postlarvae of the marine shrimp Penaeus (Penaeus) monodon and Penaeus (Fenneropenaeus) indicus in India. The latter report stimulated us to re-examine specimens from Asian shrimp farms that had experienced high mortality in Penaeus (Litopenaeus) vannamei with white muscles but tested negative for infectious myonecrosis virus (IMNV) and Penaeus vannamei nodavirus (PvNV) by RT-PCR analysis. Some of these specimens were indeed positive for MrNV by RT-PCR. Sequences of the two single-stranded RNA fragments in the MrNV genome were amplified from these P. vannamei specimens and found to share ~ 97% nucleic acid sequence identity with the MrNV sequences deposited at GenBank (NC_005094 and NC_005095). Extra small virus (XSV) usually associated with MrNV, was detected in some but not all of the samples. Infectivity tests were performed by feeding P. vannamei with minced tissues from MrNV-infected M. rosenbergii. The assays were preformed at low salinity of 2 ppt and at two different water temperatures of approximately 22 °C and 28 °C. It was revealed that shrimp exhibited a higher infectivity and mortality at the lower temperature. Our findings suggested that P. vannamei is an additional species that is susceptible to MrNV and that low temperature together with low salinity of rearing water may increase the severity of infections leading to significant mortality.
... Various diagnostic methods including histopathology have been developed in different laboratories throughout the world to detect MrNV and XSV (Arcier, et al., 1999). Other methods include immunological methods (Romestand and Bonami, 2003;Qian et al., 2006), reverse transcriptase polymerase chain reaction technique (RT-PCR) (Sri Widada et al., 2003;Sahul Hameed et al., 2004;Yoganandhan et al., 2005), loop-mediated isothermal amplification (LAMP) (Pillai et al., 2006;Puthawibool et al., 2010) in-situ hybridization method (Sri Widada et al., 2003) and dot blot hybridization (Sri Widada et al., 2004). All the above studies were aimed at detecting MrNV and XSV in the samples. ...
... These diagnostic methods include traditional methods of disease diagnosis and the methods that have been established in fish, veterinary and human diagnostic laboratories. Two immunological {sandwich enzyme-linked immunosorbent assay (S-ELISA) and triple antibody enzyme-linked immunosorbent assay (TAS-ELISA)} and four genome based methods (dot-blot and in situ hybridization, single-step RT-PCR, real-time RT-PCR and LAMP) were developed by different groups for detection of MrNV and XSV (Romestand and Bonami, 2003;Sir Widada et al., 2003Yoganandhan et al., 2005Yoganandhan et al., , 2006Chappe-Bonnichon, 2006;Pillai et al., 2006;Qian et al., 2006;Hernandez-Herrera et al., 2007). No attempt has been made to assess the sensitivity of these diagnostic methods for early detection of MrNV and XSV in WTDinfected post-larvae of freshwater prawn. ...
... In the present study, whitish muscle was observed on 7 d p.i. and 100% mortality was noted on 9 d p.i. Hence, various diagnostic methods have been developed to detect MrNV and XSV in asymptomatic conditions (Romestand and Bonami, 2003;Sir Widada et al., 2003Sahul Hameed et al., 2004;Yoganandhan et al., 2005;Pillai et al., 2006;Qian et al., 2006;Puthawibool et al., 2010). In the present study, time-course infectivity experiments were carried out on post-larvae with MrNV and XSV and four diagnostic methods, namely RT-PCR, nested RT-PCR, Western blot and ELISA were assessed for their ability to detect MrNV and XSV at an early stage in the infected post-larvae of freshwater prawn. ...
White tail disease (WTD) was found to be a serious problem in hatcheries and nursery ponds of freshwater prawn (Macrobrachium rosenbergii). The causative organisms have been identified as Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV). RT–PCR and immunological techniques such as Western blot and ELISA were used for early detection of MrNV and XSV in post-larval samples obtained from time-course experiments at different time intervals. Two viruses were purified from diseased post-larvae of M. rosenbergii by a combination of low and high speed centrifugation using sucrose and CsCl gradients to raise the antisera separately. One structural protein with molecular weight of 43 kDa (CP-43) was identified from the purified preparation of MrNV, and two overlapping polypeptides of about 17 kDa (CP-17) and 16 kDa (CP-16) were found in XSV particles by SDS-PAGE. The antisera raised against CP-43 of MrNV, CP-16 and CP17 of XSV in mice were used to detect MrNV and XSV by Western blot and ELISA. Published primers specific to MrNV and XSV were used for the early detection of these viruses by RT–PCR and nested RT–PCR. The post-larval samples collected at 3 h post infection (h p.i.) showed positive for both viruses by nested RT–PCR and negative by RT–PCR, Western blot and ELISA techniques. The samples collected at 24 h p.i. and thereafter were found to be positive for MrNV and XSV by RT–PCR, ELISA and Western blot analyses.
... The initial methods used to determine infection of MrNV and XSV employed conventional RT-PCR [17,19,21,22]. Later, multiplex RT-PCR methods were introduced for simultaneous detection of MrNV and XSV [27,30]. More sensitive was a probe-based, realtime RT-PCR analysis, but the assay was performed in a two-tube format that comprised an initial cDNA synthesis step and a subsequent amplification step [31]. ...
... The first step RT-PCR was carried out as described in a previous report [30]. However, detection was performed separately for each virus. ...
... Based on our HRM multiplex RT-PCR results, no shrimp with single XSV infections were found in our tested samples and this suggests that earlier reports showing some shrimp samples positive for XSV but not MrNV [17,30] may have been examples of MrNV loads too low to be detected by the methods used. It has been shown previously with low viral loads of another shrimp virus [6,23] that 5 serially diluted, parallel samples near the detection limit often gave variable detection results (e.g., a range of 1e4 positive out of 5 reactions) when PCR was combined with agarose gel electrophoresis. ...
In this work, a probe-free, multiplex RT-PCR was combined with high resolution melt (HRM) analysis for the simultaneous detection of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) infection in the freshwater prawn Macrobrachium rosenbergii. This first application of HRM multiplex RT-PCR in shrimp reveals a new potential for rapid and sensitive detection of multiple pathogens. In addition, sequence variation in XSV could be observed from the high resolution melt peaks, as confirmed by sequence analysis. In 19 field samples of the freshwater prawn M. rosenbergii the technique revealed samples negative for both viruses, positive for both viruses or positive for MrNV alone. No sample was found positive for XSV alone. Comparison of these results to those obtained using the same samples in analysis by traditional nested RT-PCR combined with gel electrophoresis revealed that HRM multiplex RT-PCR was more sensitive. Thus, the latter technique allows for rapid and sensitive, simultaneous detection of MrNV and XSV and also has the potential to be adapted for simultaneous detection of other mixed viral infections in shrimp.
... One of the most serious viral threats to M. rosenbergii culture is Macrobrachium rosenbergii nodavirus (MrNV) that causes white tail disease (WTD). This disease was first discovered in Guadeloupe Island [3] and subsequently in Taiwan [4], China [5], India [6], Thailand [7], and Australia [8]. MrNV is a non-enveloped, icosahedron with 26-27 nm diameter composed of a nucleocapsid bearing two positive single-stranded RNA genomes (RNA-1 and RNA-2). ...
... However, experimental transmission of MrNV revealed that the virus failed to cause mortality in adult prawns [6]. As there is no cure for WTD infected prawns, preventive procedures have been implemented such as screening of brood stock and PL, and good farm management [7,12,13]. Understanding the prawn's immune response specifically to WTD may also provide bio-rationale targets to help contain and restrict disease outbreak. ...
Background:
Macrobrachium rosenbergii, is one of a major freshwater prawn species cultured in Southeast Asia. White tail disease (WTD), caused by Macrobrachium rosenbergii nodavirus (MrNV), is a serious problem in farm cultivation and is responsible for up to 100% mortality in the post larvae stage. Molecular data on how M. rosenbergii post-larvae launches an immune response to an infection with MrNV is not currently available. We therefore compared the whole transcriptomic sequence of M. rosenbergii post-larvae before and after MrNV infection.
Results:
Transcriptome for M. rosenbergii post-larvae demonstrated high completeness (BUSCO Complete: 83.4%, fragmentation: 13%, missing:3.3%, duplication:16.2%; highest ExN50 value: 94%). The assembled transcriptome consists of 96,362 unigenes with N50 of 1308 bp. The assembled transcriptome was successfully annotated against the NCBI non-redundant arthropod database (33.75%), UniProt database (26.73%), Gene Ontology (GO) (18.98%), Evolutionary Genealogy of Genes: Non-supervised Orthologous Groups (EggNOG) (20.88%), and Kyoto Encyclopedia of Genes and Genome pathway (KEGG) (20.46%). GO annotations included immune system process, signaling, response to stimulus, and antioxidant activity. Differential abundance analysis using EdgeR showed 2413 significantly up-regulated genes and 3125 significantly down-regulated genes during the infection of MrNV.
Conclusions:
This study reported a highly complete transcriptome from the post-larvae stage of giant river prawn, M. rosenbergii. Differential abundant transcripts during MrNV infection were identified and validated by qPCR, many of these differentially abundant transcripts as key players in antiviral immunity. These include known members of the innate immune response with the largest expression change occurring in the M. rosenbergii post-larvae after MrNV infection such as antiviral protein, C-type lectin, prophenol oxidase, caspase, ADP ribosylation factors, and dicer.
... White tail disease (WTD) caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) is a major cause of 100% mortality in post-larvae (PL) but not in adult freshwater prawn M. rosenbergii [1]. MrNV was first reported in the French West Indies [2] and thereafter in China [3], India [4,5], Taiwan and Thailand [6]. MrNV is a small, icosahedral, non-enveloped virus. ...
... B2 protein is encoded in the sub-genomic RNA3 and its function is a suppressor in post-transcriptional gene silencing whereas the RNA2 (1.2 kb) encodes a capsid protein in size of 43 kDa [7,8]. There are several methods for detection of MrNV such as RT-PCR, multiplex RT-PCR [6], TaqMan realtime RT-PCR, in situ hybridization, dot blot hybridization [9], double antibody sandwich enzyme-linked immunosorbant assay [10], monoclonal antibodies based assay [11], loop-mediated isothermal amplification [12,13], and high resolution melt duplex RT-PCR [14]. ...
Apoptosis is an essential immune response to protect invertebrates from virus infected cells. In shrimp, virus infection has been reported to induce apoptosis. Macrobrachium rosenbergii (Mr) was considered to be a disease-resistant host when compared to penaeid shrimps. Caspase-3 was classified as an executioner caspase which played a key role in virus-induced apoptosis. In this study, an effector caspase gene of M. rosenbergii (Mrcasp) was cloned and characterized. The open reading frame (ORF) of Mrcasp was 957 nucleotide encoding 318 amino acid with a deduced molecular mass of 35.87 kDa. RT-PCR analysis showed the presence of Mrcasp in all examined tissues. The phylogenetic tree indicated that Mrcasp was closely related with caspase 3 of shrimp. The functions of the Mrcasp, B2 and capsid proteins of Macrobrachium rosenbergii nodavirus (MrNV) were assayed in Sf-9 cells. The results showed that Mrcasp induce apoptotic morphology cells; however, capsid protein of MrNV could inhibit apoptotic cells whereas B2 could neither induce nor inhibit apoptotic cells by DAPI staining. The protein interaction between Mrcasp and viral MrNV structure revealed that Mrcasp did not bind to B2 or capsid protein whereas B2 and capsid proteins could bind directly to each other. This study reported a novel sequence of a full-length Mrcasp and its functional studies indicated that Mrcasp could induce apoptotic cells. Our data is the first report demonstrating the direct protein-protein interaction between capsid protein and B2 protein of MrNV.
Copyright © 2015. Published by Elsevier Ltd.
... The protocol for the RT-PCR for detection of MrNV/XSV developed by Sri Widada et al. [24] and Sahul Hameed et al. [20,21] is recommended for all situations. MrNV and XSV can be detected by RT-PCR separately using a specific set of primers or these two viruses can be detected simultaneously using a single-tube one-step multiplex RT-PCR [32,36]. Nested RT-PCR (nRT-PCR) is also available and recommended for screening brood stock and seed [26]. ...
... However, proper preventive measures, such as screening of brood stock and PL, and good management practices may help to prevent WTD in culture systems. As the life cycle of M. rosenbergii is completed under controlled conditions, specific pathogen free (SPF) brood stock and PL can be produced by screening using sensitive diagnostic methods such as reverse-transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) [16,24,36]. Hayakijkosol and Owens [4] reported a sequence specific dsRNA against protein B2 produced RNAi that was able to functionally prevent and reduce mortality in WTD-infected redclaw crayfish. ...
Macrobrachium rosenbergii is the most important cultured freshwater prawn in the world and it is now farmed on a large scale in many countries. Generally, freshwater prawn is considered to be tolerant to diseases but a disease of viral origin is responsible for severe mortalities in larval, post-larval and juvenile stages of prawn. This viral infection namely white tail disease (WTD) was reported in the island of Guadeloupe in 1995 and later in Martinique (FrenchWest Indies) in Taiwan, the People's Republic of China, India, Thailand, Australia and Malaysia. Two viruses, Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus-like particle (XSV) have been identified as causative agents of WTD. MrNV is a small icosahedral non-enveloped particle, 26-27 nm in diameter, identified in the cytoplasm of connective cells. XSV is also an icosahedral virus and 15 nm in diameter. Clinical signs observed in the infected animals include lethargy, opaqueness of the abdominal muscle, degeneration of the telson and uropods, and up to 100 % within 4 days. The available diagnostic methods to detect WTD include RT-PCR, dot-blot hybridization, in situ hybridization and ELISA. In experimental infection, these viruses caused 100 % mortality in post-larvae but failed to cause mortality in adult prawns. The reported hosts for these viruses include marine shrimp, Artemia and aquatic insects. Experiments were carried out to determine the possibility of vertical transmission of MrNV and XSV in M. rosenbergii. The results indicate that WTD may be transferred from infected brooders to their offspring during spawning. Replication of MrNV and XSV was investigated in apparently healthy C6/36 Aedes albopictus and SSN-1 cell lines. The results revealed that C6/36 and SSN-1cells were susceptible to these viruses. No work has been carried out on control and prevention of WTD and dsRNA against protein B2 produced RNAi that was able to functionally prevent and reduce mortality in WTD-infected redclaw crayfish.
... Viral inoculum was prepared following the method described by Ravi et al. (2009) with some minor modi- fications. Infected samples were tested for presence of MrNV by RT-PCR using primers designed by Yoganandhan et al. (2005). Posi- tive samples were homogenized in a sterile homogenizer. ...
... Several RT-PCR assays have been developed for the detection of PvNV and MrNV of different geographical region ( Hayakijkosol and Owens, 2011;Owens et al., 2009;Sri Widada et al., 2003;Yoganandhan et al., 2005;Sahul Hameed et al., 2004;Tang et al., 2007). Sequence variation within MrNV of different geographical isolates and wide variation between MrNV and PvNV may result in mismatches between the primers and the sequence of their bind- ing site which causes variation in sensitivity or even failure of detection. ...
... The pathogenesis of WTD in Macrobrachium rosenbergii was initially reported as virus; later, it was found that Macrobrachium rosenbergii nodavirus (MrNV) (Arcier et al. 1999, Sahul Hameed et al. 2004) and extra small virus (XSV) (Qian et al. 2003, Sahul Hameed et al. 2004Yoganandhan et al. 2005) were the causative agents for the disease. The first case of WTD was reported from Island of Guadeloupe in 1995, then in Martinique (French West Indies) (Arcier et al. 1999), Taiwan (Tung et al. 1999, The People's Republic of china (Qian et al. 2003) and India (Sahul Hameed et al. 2004).WTD is mainly diagnosed by the opaqueness of the abdominal muscle, degeneration of telson and uropods, and 100% mortality within a span of 4 days (Arcier et al. 1999;Sahul Hameed et al. 2004). ...
... In confirmation of white muscle disease by RT-PCR, samples were positive to MrNV and XSV, but sample was positive to XSV and negative to MrNV. Similar observations were done by Yoganandhan et al. (2005). ...
Abstract
A 60-day feeding trial was conducted to evaluate the effect of dietary nucleotide on growth, survival, immunity and resistance to white muscle disease and Aeromonas hydrophila infection in freshwater prawn (Macrobrachium rosenbergii). The nucleotide was supplemented at 0, 1.5, 2.25 and 3.0 g/kg diet. The test diets were fed for 60 days in triplicate groups of prawns, which had initial weight of 0.27 g. At the end of the feeding trial, growth was recorded and non-specific immune parameters, such as, prophenol oxidase activity, superoxide anion production, total haemocyte count and total serum protein, were studied in haemolymph samples. Phenol oxidase activity and superoxide anion production were significantly (P < 0.05) higher in prawns fed nucleotide-based diets. Total haemocyte count and haemolymph were higher (P < 0.05) in prawns fed nucleotide-based diets. The relative per cent survival of prawn after the challenge test against white muscle disease was significantly (P < 0.05) higher in prawn fed nucleotide-incorporated diets. However, there was no effect of nucleotide supplementation on growth and survival of prawn.
... An initial need for development of such stocks is the preparation of a list of excludable pathogens for screening of the founder stocks. Of particular importance for this list would be viral pathogens such as Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) that are causally related to white tail disease , Sahul Hameed et al. 2004, Bonami et al. 2005, Yoganandhan et al. 2006) and result in severe mortality in hatcheries. These viruses may be transmitted vertically from naturally infected broodstock to larvae (Sudhakaran et al. 2007) or via reservoir carriers that may include other shrimp (Sudhakaran et al. 2006a,b) or even aquatic insects (Sudhakaran et al. 2008). ...
... For successful specific pathogen free (SPF) stock development, diagnostic methods would be required for all of the pathogens included in any specific list of pathogens for M. rosenbergii. For this species, highly sensitive RT-PCR methods are available for MrNV and XSV (Romestand & Bonami 2003, Sri Widada et al. 2003, Yoganandhan et al. 2005, Pillai et al. 2006). However, for other viral pathogens, such as the parvo-like virus described above, no molecular methods have been developed. ...
A survey of cultivated giant freshwater prawns Macrobrachium rosenbergii from Thailand revealed the presence of unusual spherical to ovoid inclusions in nuclei of hepatopancreas tubule epithelial cells. These began as small eosinophilic inclusions that became more basophilic as they increased in size. They were present in both R-cells and E-cells but were largest and deeply basophilic only in the E-cells. Confocal laser microscopy revealed that stained nucleic acid fluorescence from the inclusions was lost by treatment with DNase I specific for double- and single-stranded DNA and also lost or reduced by treatment with mungbean nuclease specific for single-stranded nucleic acids. Transmission electron microscopy (TEM) revealed that the inclusions contained tightly packed, unenveloped, viral-like particles of approximately 25 to 30 nm diameter, resembling those produced by shrimp parvoviruses. However, PCR, in situ hybridization and immunohistochemical tests for shrimp parvoviruses previously reported from Thailand were all negative. These results suggested that the inclusions contained a parvo-like virus, not previously reported from M. rosenbergii in Thailand.
... Now there is XSV specific detection based on genome analysis (Widada et al., 2004). Simultaneous detection in one tube for WTD (MrNV-XSV) has also been tried (Yonganandhan et al., 2005;Tripathy et al., 2006). Qualitative detection could be conducted by using Tagman probe for Real Time or Quantitative Polymerase Chain Reaction (q-PCR) (Zhang et al., 2006). ...
Mass mortality of giant freshwater prawn (Macrobrachium rosenbergii de Man) in grow-out farmers occurred in early February 2012 at Instalation Coastal of Aquaculture Samas, Bantul, D.I. Yogyakarta. The clinical sign of shrimp was whitish coloration on abdominal and tail muscle. The sympton was the same as in other cases of mortality caused by prawn Macrobrachium rosenbergii nodavirus (MrNV) and Extra Small Virus (XSV). Prawn samples were diagnosed by standard protocols Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) using specific primers and histopathology analysis. The result showed that all samples indicated positive 13/15 the MrNV and 5/15 positive XSV, and there were 4/15 positive samples both (MrNV and XSV). Analysis of histopathology showed that damaged muscle was indicated by the presentation of necrotic tissues with nuclear pyknosis or degeneration of muscle in infected tissues. Based on diagnosis by RT-PCR and histopathological, mass mortality of the giant freshwater prawn in Indonesia is determined to be caused by “white muscle disease (WMD)/white tail disease (WTD)”.
... To avoid having to carry out two RT-PCR reactions, one for each virus, we developed a modified method, one-step multiplex RT-PCR (mRT-PCR), to simultaneously detect both in a single tube (Yoganandhan et al., 2005). These concurred with bands obtained in separate RT-PCR assays, one for each virus. ...
Macrobrachium rosenbergii, a global and economically important cultured freshwater prawn, is farmed on a large scale in many countries. Compared to penaeid shrimps, M. rosenbergii is a moderately disease-resistant species. However, viruses such as Macrobrachium hepatopancreatic parvo-like virus (MHPV), Macrobrachium muscle virus (MMV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), white spot syn- drome virus (WSSV), Macrobrachium rosenbergii nodavirus (MrNV), and extra small virus-like particle (XSV) have been reported and are responsi- ble for economic losses to freshwater prawn culture. MrNV and XSV, the causes of white tail disease (WTD), have been reported as dangerous viruses to M. rosenbergii, resulting in 100% mortality in postlarvae and juve- niles within five days of infection. Clinical signs of WTD include lethargy and opaqueness of the abdominal muscle. Various aspects of WTD are dis- cussed in this paper, including tissue tropism of the causative viruses, host range, virus structure, vertical transmission, pathogenicity, and the possibil- ity of multiplying MrNV and XSV in a mosquito cell line.
... With the developed duplex PCR, detection and differentiation of WSSV and EHP could be achieved in a single reaction. Multiplex PCR and RT-PCR have been developed for simultaneous detection of viral pathogens of shrimp and prawn (Khawsak, Deesukon, Chaivisuthangkura, & Sukhumsirichart, 2008;Natividad et al., 2006;Sibonga, Geduspan, & Caipang, 2013;Xie et al., 2007;Yang et al., 2006;Yoganandhan, Sri Widada, Bonami, & Hameed, 2005). In conclusion, the duplex PCR protocol developed in the present study for simultaneous detection of WSSV and EHP, which can be completed within 8 hr, provides significant saving time, cost and materials in comparison with conventional PCR assay. ...
White leg shrimp, Penaeus vannamei, were collected on a monthly basis from grow‐out ponds located at Tamil Nadu and Andhra Pradesh states along the east coast of India for screening of viral and other pathogens. Totally 240 shrimp samples randomly collected from 92 farms were screened for white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV), infectious myonecrosis virus (IMNV) and Enterocytozoon hepatopenaei (EHP). The number of shrimp collected from shrimp farms ranged from 6 to 20 based on the body weight of the shrimp. All the shrimp collected from one farm were pooled together for screening for pathogens by PCR assay. Among the samples screened, 28 samples were WSSV‐positive, one positive for IHHNV and 30 samples positive for EHP. Among the positive samples, four samples were found to be positive for both WSSV and EHP, which indicated that the shrimp had multiple infections with WSSV and EHP. This is the first report on the occurrence of multiple infections caused by WSSV and EHP. Multiplex PCR (m‐PCR) protocol was standardized to detect both pathogens simultaneously in single reaction instead of carrying out separate PCR for both pathogens. Using m‐PCR assay, naturally infected shrimp samples collected from field showed two prominent bands of 615 and 510 bp for WSSV and EHP, respectively.
... Macrobrachium rosenbergii PL and P. vannamei exhibiting whitish muscle syndrome were collected from a hatchery in Ratchaburi Province, Thailand and screened for MrNV infection by RT-PCR with specific primers targeted to RNA2 (Yoganandhan et al., 2005). Total RNA was extracted from infected shrimp using TriPure isolation reagent (Roche, Mannheim, Germany) following the manufacturer's instructions. ...
Macrobrachium rosenbergii nodavirus (MrNV) is usually accompanied by extra small virus (XSV) in natural outbreaks of white tail disease (WTD) in the giant river prawn Macrobrachium rosenbergii. Testing the virulence of MrNV alone has been problematic due to the difficulty in completely separating XSV from MrNV by viral purification steps from naturally infected shrimp. However, based on reports of natural M. rosenbergii specimens from WTD outbreak ponds that were positive for MrNV but negative for XSV led us to hypothesize that MrNV alone might cause WTD. To test this hypothesis, we prepared the two, complete genomic RNA fragments (RNA1 and RNA2) of MrNV from cDNA clones and used these to transfect Sf9 cells that subsequently showed cellular changes, including cell swelling, syncytial cell formation, and development of cytoplasmic inclusions within 72 h post-transfection. Replication of RNA1 and RNA2 increased in the transfected cells and transmission electron microscopy of the cell lysates revealed the presence of icosahedral viral-like particles that were 40–50 nm in diameter. When naïve Sf9 cells were inoculated with the cell lysate, the newly infected cells showed cellular changes and produced strong immunoreactivity against MrNV capsid protein indicating the infectious nature of the cell lysate. When the lysates were injected into the whiteleg shrimp Penaeus vannamei, MrNV RNA replication in the shrimp was followed by morality accompanied by typical MrNV lesions that gave possible positive immunohistochemical reactions for the MrNV capsid protein. Treatment of the Sf9 cells with azidothymidine triphosphate (AZT) prior to transfection significantly increased viral RNA synthesis and pathogenicity when compared with untreated, transfected cells. Using this model to produce infectious MrNV without XSV contamination proves that MrNV alone can be lethal to shrimp and it opens the way to further investigate the molecular basis of MrNV pathogenesis, and to develop antiviral strategy to control white tail disease.
... Its genome is formed by two pieces of ss RNA of 2.90 and 1.26 kb, respectively. (Sri Widada et al. 2003; Yoganandhan, Sri Widada, Bonami & Sahul Hameed 2005). Satellite virus-like particles (named XSV) are 15 nm in diameter with an ss RNA genome of 0.8–0.9 kb coding only for its capsid protein but lacking the polymerase gene (RdRp); thus, XSV is unable to replicate alone. ...
Macrobrachium rosenbergii WTD MrNV XSV Penaeus monodon Penaeus indicus Host White tail disease (WTD) caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small viruses (XSV) is a major problem. It is responsible for severe mortality in post-larvae of M. rosenbergii in the hatcheries and nurseries. These viruses have a wide host range including marine shrimp. Recently, WTD has been observed in hatchery reared post-larvae of marine shrimp (Penaeus monodon and P. indicus). Clinical signs observed in these animals were found to be similar to those found in the post-larvae of M. rosenbergii. The infected post-larvae showed positive for MrNV and XSV by RT-PCR. The inoculum prepared from these infected post-larvae caused 100% mortality in the post-larvae of freshwater prawn.
... Its genome is formed by two pieces of ss RNA of 2.90 and 1.26 kb, respectively. (Sri Widada et al. 2003;Yoganandhan, Sri Widada, Bonami & Sahul Hameed 2005). Satellite virus-like particles (named XSV) are 15 nm in diameter with an ss RNA genome of 0.8-0.9 kb coding only for its capsid protein but lacking the polymerase gene (RdRp); thus, XSV is unable to replicate alone. ...
Macrobrachium rosenbergii WTD MrNV XSV Penaeus monodon Penaeus indicus Host White tail disease (WTD) caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small viruses (XSV) is a major problem. It is responsible for severe mortality in post-larvae of M. rosenbergii in the hatcheries and nurseries. These viruses have a wide host range including marine shrimp. Recently, WTD has been observed in hatchery reared post-larvae of marine shrimp (Penaeus monodon and P. indicus). Clinical signs observed in these animals were found to be similar to those found in the post-larvae of M. rosenbergii. The infected post-larvae showed positive for MrNV and XSV by RT-PCR. The inoculum prepared from these infected post-larvae caused 100% mortality in the post-larvae of freshwater prawn.
... Several diagnostic methods of WTD using polymerase chain reaction (PCR) and reverse transcriptase-PCR (RT-PCR) methods have been reported (Sri-Widada, Durand, Cambournac, Qian, Shi, Dejonghe, Richard & Bonami 2003;Sahul-Hameed, Yoganandhan, Sri-Widada & Bonami 2004b;Sri-Widada, Richard, Cambournac, Shi, Qian & Bonami 2004). A one-step multiplex RT-PCR for simultaneous detection of MrNV and XSV in naturally and experimentally infected prawns (Yoganandhan, Sri-Widada, Bonami & Sahul-Hameed 2005), and a cost-effective field level diagnostic kit for detection of MrNV by the loop-mediated isothermal amplification technique (Pillai, Bonami & Sri-Widada 2006) have also been developed. However, an organized disease diagnosis and surveillance system similar to that existing in the case of marine shrimp farming, has yet to be established in freshwater prawn farming in India, because the diagnostic laboratories still focus on marine shrimps, and the rapid detection kits for freshwater prawn diseases have not become popular. ...
Freshwater prawn production in India that includes farming and wild capture of the giant freshwater prawn, Macrobrachium rosenbergii and the monsoon river prawn, M. malcolmsonii has increased steadily since 1999 reaching a peak output of 42 780 t in 2005, but then declined to 6568 t in 2009–2010. Stunted growth and diseases in ponds because of poor seed quality and the broodstock which had been inbred over several generations; pond water quality issues; and increased cost of production on account of feed, labour and the mandatory certification requirements are suggested to be some of the factors leading to the production declines. While majority of the output occurs in Andhra Pradesh, single crop paddy–prawn production systems in the low-lying fields of Kerala have helped gradual transformation to a sustainable, organic mode of farming of both rice and prawns, suitable for other states of India. Although the trends by June 2011 indicate that the sector is set to a revival, future prospects of freshwater prawn farming in India will also depend on the expansion of whiteleg shrimp Litopenaeus vannamei that was introduced recently in India and provided a more profitable opportunity for farming.
... WTD causes significant mortality in hatchery-and nursery-reared postlarvae [94] . The disease was first reported in Guadeloupe Island (French West Indies) in 1997 [95] and was later reported in China [96] , India [97] , Thailand [98] , Taiwan [99] and Australia [100] . MrNV is a nonenveloped, icosahedral virus with a diameter of 26-27 nm and a genome comprised of two pieces of positive-sense ssRNA. ...
Among shrimp viral pathogens, white spot syndrome virus (WSSV) and yellow head virus (YHV) are the most lethal agents, causing serious problems for both the whiteleg shrimp, Penaeus (Litopenaeus) vannamei, and the black tiger shrimp, Penaeus (Penaeus) monodon. Another important virus that infects P. vannamei is infectious myonecrosis virus (IMNV), which induces the white discoloration of affected muscle. In the cases of taura syndrome virus and Penaeus stylirostris densovirus (PstDNV; formerly known as infectious hypodermal and hematopoietic necrosis virus), their impacts were greatly diminished after the introduction of tolerant stocks of P. vannamei. Less important viruses are Penaeus monodon densovirus (PmDNV; formerly called hepatopancreatic parvovirus), and Penaeus monodon nucleopolyhedrovirus (PemoNPV; previously called monodon baculovirus). For freshwater prawn, Macrobrachium rosenbergii nodavirus and extra small virus are considered important viral pathogens. Monoclonal antibodies (MAbs) specific to the shrimp viruses described above have been generated and used as an alternative tool in various immunoassays such as enzyme-linked immunosorbent assay, dot blotting, Western blotting and immunohistochemistry. Some of these MAbs were further developed into immunochromatographic strip tests for the detection of WSSV, YHV, IMNV and PemoNPV and into a dual strip test for the simultaneous detection of WSSV/YHV. The strip test has the advantages of speed, as the result can be obtained within 15 min, and simplicity, as laboratory equipment and specialized skills are not required. Therefore, strip tests can be used by shrimp farmers for the pond-side monitoring of viral infection.
... Several tests have been reported for detecting WTD which include dot-blot hybridization, in situ hybridization and reverse-transcriptase polymerase chain reaction (RT-PCR) [11], loop-mediated isothermal amplification (LAMP) [4], sandwich enzyme-linked immunosorbent assay (S-ELISA) [7] and triple antibody sandwich enzymelinked immunosorbent assay (TAS-ELISA) [6] for MrNV; dot-blot hybridization and RT-PCR for XSV [12] and onestep multiplex RT-PCR [13,16] for the detection of both MrNV and XSV. Sahul Hameed et al. [9] developed either against the whole virus particles or against the capsid proteins obtained by electroelution from SDS-PAGE separated viral proteins. ...
Macrobrachium rosenbergii nodavirus along with a satellite virus, extra small virus (XSV) causes white tail disease (WTD) in the giant freshwater prawn M. rosenbergii. Infected M. rosenbergii postlarvae were collected from a hatchery in Kakinada, Andhra Pradesh. The gene coding the capsid protein of XSV was cloned in a bacterial expression vector pRSET A and the recombinant protein was expressed in Escherichia coli BL21(DE3)pLysS cells. The recombinant protein was purified by Nickel affinity chromatography. Polyclonal antibodies were produced in mice against the recombinant protein and the antibodies reacted specifically with the recombinant protein and XSV in WTD-infected tissues. This is the first report of detection of XSV using antibodies against recombinant capsid protein.
... Its genome is formed by two pieces of ss RNA of 2.90 and 1.26 kb, respectively. (Sri Widada et al. 2003;Yoganandhan, Sri Widada, Bonami & Sahul Hameed 2005). Satellite virus-like particles (named XSV) are 15 nm in diameter with an ss RNA genome of 0.8-0.9 kb coding only for its capsid protein but lacking the polymerase gene (RdRp); thus, XSV is unable to replicate alone. ...
This study evaluated the possible use of the fish SSN-1 cell line to investigate the development of Macrobrachium rosenbergii nodavirus (MrNV). Cells were incubated with viral particles and cytopathic effects were observed. De novo synthesis of viral capsid proteins was shown by immuno-fluorescence labelling and a sandwich ELISA test. Viral genomic replication was demonstrated by RT-PCR using primers specific to RNA-1 as well as by quantitative RT-PCR (RT-qPCR). Using electron microscopy, only a few empty particles were observed and attempts to isolate complete infectious particles or to re-infect healthy cells (second passage) were unsuccessful. As complete viral particles were rarely observed, it appeared that defaults in MrNV virogenesis might arise resulting in the formation of scarce and non-infectious particles. SSN-1 cells were found to be partially permissive to MrNV infection that induced cell lysis, but key elements for viral infection were lacking such as regulatory factors for gene replication or post-translational modifications.
... 3 2003), (Bonami et al., 2005). Recently, it has been reported that the mixed MrNV/XSV infection can also cause WTD with high mortality in larvae of the brackishwater shrimp Penaeus (Penaeus) monodon and Penaeus (Fenneropenaeus) indicus (Ravi et al., 2009), although not in juveniles of the same species (Sudhakarana et al., 2006). ...
Whitening of muscle tissue in farmed whiteleg shrimp Penaeus (Litopenaeus) vannamei can result from stress-induced muscle cramps and from viral infections caused by infectious myonecrosis virus (IMNV) or Penaeus vannamei nodavirus (PvNV). A similar viral-induced whitening of muscles can be caused in the river prawn Macrobrachium rosenbergii and in larvae of the penaeid shrimp Penaeus (Penaeus) monodon and Penaeus (Fenneropenaeus) indicus by a mixed infection with Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV). Here we describe mixed infections of IMNV and MrNV in juvenile cultivated P. vannamei from Indonesia, detected by nested RT-PCR and immunohistochemical analysis. Muscle lesions in the dually-infected shrimp gave positive immunohistochemical reactions for both IMNV and MrNV, while connective tissue in the same samples gave positive reactions for MrNV only, indicating some differences in tissue specificity between the two viruses. Although it is not known whether the dual infections are more lethal to shrimp than single IMNV infections, this is possible since earlier work has shown that MrNV alone can increase mortality of P. vannamei under stress.
... Several diagnostic methods of WTD using PCR have been reported. A one-step multiplex reverse transcriptasepolymerase chain reaction (RT-PCR) was developed for simultaneous detection of MrNV and XSV in naturally and experimentally infected shrimp (Sahul-Hameed et al. 2004, Yoganandhan et al. 2005). Lessons from shrimp farming (Kutty 2005), which tread similar paths a decade earlier, clearly point out the need for more vigilance in prawn health management for ensuring sustainable prawn farming. ...
Aquaculture contributed 38 percent of the global fisheries pro-duction of 159 million t in 2004. Asian aquaculture provided the bulk (91 percent) of the global aquaculture production, and 76 percent of that came from one country, China (FAO 2006). The growing importance of aquacul-ture in overcoming production limits of capture fisheries can be judged from the fact that China's 2004 production through aqua-culture was about 70 percent of its total fisheries production. South Asia contributed 7.5 per-cent of total world aquaculture production in 2004, most of which came from India (5.4 percent) and Bangladesh (2 percent, Figures 1 and 2). The contribution of South Asia in world farmed production of crustaceans, marine shrimp and freshwater shrimp is shown in Fig-ure 3, which indicates the relative importance of freshwater shrimp in the aquaculture and economy of the sub-region. Crustaceans in general and shrimp in particular, are high value aquatic food commodities globally (Table 1). South Asia, specifically the Indian subconti-nent, produced 56,091 t of farmed freshwater shrimp in 2004, which came from India and Bangladesh (Figure 4). Actual South Asian pro-duction could have been higher be-cause freshwater shrimp farming ex-ists on a smaller scale in Sri Lanka and Pakistan as well. Nepal has just begun their farming trials with locally produced freshwater shrimp postlar-vae. Farming developments in India, Bangladesh, Sri Lanka, Pakistan and Fig. 1. Map of South Asia showing country-wise species and production details of freshwater shrimp farming. in the wake of the near collapse of marine shrimp farming in the late nineties. M. rosenbergii cul-ture has progressed rapidly since 1999. Farmed freshwater shrimp production and the area being farmed increased steadily from 1999 to reach 42,780 t and 43,395 ha in 2006 (Figure 5), 87 percent of which came from Andhra Pradesh State. West Bengal was a distant second (9 percent), fol-lowed by Orissa, Tamil Nadu, Kerala and Maharashtra. Com-mercial farming is almost en-tirely M. rosenbergii. The small production of M. malcolmsonii, 4 is based mainly on capture-based farming (Kutty et al. 2000). Freshwater shrimp exports in all product categories in 2004-2005 was 9,401 t (value: US$84 million). The 2005-2006 export levels were less (6,320 t, US$ 57 million). The pro-portion of exported quantity to to-tal annual production also decreased from 24 to 14 percent, possibly be-cause of increased domestic con-sumption, global competition and trade barriers. Domestic consump-tion has been increasing steadily and is now about 50 percent of the total production. India has 71 hatcheries with a to-tal annual capacity of about 1,800 million postlarval M. rosenbergii. A few experimental hatcheries for M. malcolmsonii also exist, but consider-able stocking of that species in ponds and open waters is based on the capture of wild juveniles, mainly in Andhra Pradesh and Tamil Nadu (New et al. 2000). Over 60 percent of the hatch-eries are located in Andhra Pradesh. Most of them are coastal multi-species Macrobrachium malcolmsonii from River Godavary, Andhra Pradesh. (Photo by K.R. Salin)
... It can be applied for routine health monitoring, early virus detection, studying virus-host interaction, detection of carriers and screening of broodstock (Sri Widada et al., 2003;Sahul Hameed et al., 2004b). Previously reported detection limits were 3000 virus particles by RT-PCR (Sri Widada et al., 2003), 0.25 fg of total RNA by RT-PCR (Sahul Hameed et al., 2004a), 0.01 fg of total viral RNA corresponding approximately to four viral particles by nested RT-PCR (Anonymous, 2004), and 2.5 fg of total RNA by single tube one step MRT-PCR (Yoganandhan et al., 2005). Similarly, an RT-PCR method for XSV detection had a sensitivity of 5 fg of viral RNA . ...
A multiplex reverse transcriptase polymerase chain reaction (MRT-PCR) assay was developed and applied to the detection of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) associated with field and hatchery outbreaks of white tail disease (WTD) in M. rosenbergii. Primers targeted regions of the capsid protein genes of the two viruses and produced amplicons of 1136 and 447 bp, for MrNV and XSV, respectively, with sensitivity to the level of 1 fg of total RNA for both the viruses. Assays could be completed within 7 to 9 h after samples were received. This is of great importance for epidemiological analysis and screening of large numbers of samples. MRT-PCR assay on samples collected from different hatcheries and farms of Nellore, India revealed the presence of both the viruses in all. Experimental infections produced typical clinical signs of white muscle in juveniles and mortality up to 20%. MrNV could be detected up to 1 month in surviving prawns, both by histology and RT-PCR. Sequence analysis of capsid protein gene amplicons of XSV revealed minor variations. By contrast, sequence analysis of MrNV amplicons showed no variation in amino acid sequence of the capsid protein gene.
... Genome-based detection methods with high specificity and high sensitivity for detection of MrNV include dot blot hybridization, in situ hybridization (Sri Widada et al. 2003) and 1-step RT-PCR in the form of single tests for MrNV or XSV (Sri Widada et al. 2003, Sahul Hameed et al. 2004 or multiplex tests for both viruses (Yoganandhan et al. 2005, Tripathy et al. 2006. A 2-step real-time RT-PCR method (Zhang et al. 2006) and a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method (Pillai et al. 2006, Puthawibool et al. 2010) have also been described. ...
The gene encoding the capsid protein of Macrobrachium rosenbergii nodavirus (MrNV) was cloned into pGEX-6P-1 expression vector and then transformed into the Escherichia coli strain BL21. After induction, capsid protein-glutathione-S-transferase (GST-MrNV; 64 kDa) was produced. The recombinant protein was separated using SDS-PAGE, excised from the gel, electro-eluted and then used for immunization for monoclonal antibody (MAb) production. Four MAbs specific to the capsid protein were selected and could be used to detect natural MrNV infections in M. rosenbergii by dot blotting, Western blotting and immunohistochemistry without cross-reaction with uninfected shrimp tissues or other common shrimp viruses. The detection sensitivity of the MAbs was 10 fmol µl-1 of the GST-MrNV, as determined using dot blotting. However, the sensitivity of the MAb on dot blotting with homogenate from naturally infected M. rosenbergii was approximately 200-fold lower than that of 1-step RT-PCR. Immunohistochemical analysis using these MAbs with infected shrimp tissues demonstrated staining in the muscles, nerve cord, gill, heart, loose connective tissue and inter-tubular tissue of the hepatopancreas. Although the positive reactions occurred in small focal areas, the immunoreactivity was clearly demonstrated. The MAbs targeted different epitopes of the capsid protein and will be used to develop a simple immunoassay strip test for rapid detection of MrNV.
... Among the samples analyzed, many MrNV-positive samples were also positive for XSV. In some cases, XSV was however absent, and curiously in a few samples MrNV was undetected while XSV presence was observed (Fig. 12) (Sri Widada and Bonami, unpublished data;Yoganandhan et al., 2005Yoganandhan et al., , 2006. This raises the question of the viral development of XSV within the infected cells. ...
The giant freshwater prawn Macrobrachium rosenbergii is cultivated essentially in Southern and South-eastern Asian countries such as continental China, India, Thailand and Taiwan. To date, only two viral agents have been reported from this prawn. The first (HPV-type virus) was observed by chance 25 years ago in hypertrophied nuclei of hepatopancreatic epithelial cells and is closely related to members of the Parvoviridae family. The second, a nodavirus named MrNV, is always associated with a non-autonomous satellite-like virus (XSV), and is the origin of so-called white tail disease (WTD) responsible for mass mortalities and important economic losses in hatcheries and farms for over a decade. After isolation and purification of these two particles, they were physico-chemically characterized and their genome sequenced. The MrNV genome is formed with two single linear ss-RNA molecules, 3202 and 1250 nucleotides long, respectively. Each RNA segment contains only one ORF, ORF1 coding for the RNA-dependant RNA polymerase located on the long segment and ORF2 coding for the structural protein CP-43 located on the small one. The XSV genome (linear ss-RNA), 796 nucleotides long, contains a single ORF coding for the XSV coat protein CP-17. The XSV does not contain any RdRp gene and consequently needs the MrNV polymerase to replicate.
... rosenbergii) adults and post-larva were obtained from shrimp farms in Ratchaburi and Kanchanaburi Provinces in Thailand and from Myanmar in August 2006. They were positive for MrNV using a previously described RT-PCR detection method [20]. Normal whiteleg shrimp were obtained from Samutsakhon province of Thailand. ...
Loop-mediated isothermal amplification (LAMP) allows rapid amplification of nucleic acids under isothermal conditions. It can be combined with a chromatographic lateral flow dipstick (LFD) for much more efficient, field-friendly detection of MrNV. In this work, RT-LAMP was performed at 65 degrees C for 40 min, followed by 5 min for hybridization with an FITC-labeled DNA probe and 5 min for LFD resulted in visualization of DNA amplicons trapped at the LFD test line. Thus, total assay time, including 10 min for rapid RNA extraction was approximately 60 min. In addition to advantages of short assay time, confirmation of amplicon identity by hybridization and elimination of electrophoresis with carcinogenic ethidium bromide, the RT-LAMP-LFD was more sensitive than an existing RT-PCR method for detection of MrNV. The RT-LAMP-LFD method gave negative test results with nucleic acid extracts from normal shrimp and from shrimp infected with other viruses including DNA viruses [PstDNV (IHHNV), PemoNPV (MBV), PmDNV (HPV), WSSV] and RNA viruses (TSV, IMNV, YHV/GAV).
... In order to carry out a successful program for SPF stock development, it is necessary to have diagnostic tools appropriate for major pathogens that may have a negative impact on production. For M. rosenbergii, highly sensitive RT-PCR methods are available for MrNV and XSV (Romestand and Bonami, 2003;Sri Widada et al., 2003Pillai et al., 2006;Yoganandhan et al., 2005). There is also a 0168-1702/$ -see front matter © 2009 Elsevier B.V. All rights reserved. ...
Field specimens of post-larvae of the giant freshwater prawn (Macrobrachium rosenbergii) from Thailand showed hepatopancreatic tubule epithelial cells that contained central, eosinophilic inclusions within enlarged nuclei and marginated chromatin. These inclusions resembled those produced by some baculoviruses prior to formation of occlusion bodies that enclose virions in a polyhedrin protein matrix. By electron microscopy, the intranuclear inclusions contained bacilliform, enveloped virions (approximately 327+/-29nmx87+/-12nm) with evenly dense, linear nucleocapsids surrounded by trilaminar envelopes with lateral pockets containing nucleoproteinic filaments. In some cases, these were accompanied by moderately electron dense, spherical particles of approximately 20nm diameter resembling polyhedrin subunits of occlusion bodies (OB) of a bacilliform virus of the black tiger shrimp Penaeus monodon, previously reported from Thailand and called monodon baculovirus (MBV). It is currently listed by the International Committee on Taxonomy of viruses as Penaeus monodon nucleopolyhedrovirus (PemoNPV). Two polymerase chain reaction (PCR) assays for MBV gave positive results with DNA extracts prepared from M. rosenbergii samples using the hot phenol technique. One of these assays targeted the polyhedrin gene of MBV to which the resulting amplicon showed 100% sequence identity. Presence of the Penaeus monodon virus polyhedrin gene was confirmed by in situ hybridization assays and by positive immunohistochemical reactions in one sample batch. The data revealed that MBV can be found but may rarely produce polyhedrin occlusion bodies in M. rosenbergii.
... LaSudhakaran et al., 2006 ;). La RT-PCR simple ou double, et la MRT-PCR (Multiplex RT-PCR) permettant la détection simultanée des deux virus (Sri Widada et al., 2003; Yoganandhan et al., 2005 ; Tripathy S. et al., 2006) ...
Penaeid family shrimp constitute the first aquaculture product in the world in terms of commercial value. They are produced in third world countries of the sub-equatorial belt. Among the causes limiting their production, one is the presence of the WSSV (White Spot Syndrome Virus), a pathogenic agent that produces massive mortality. Our aim was to investigate the first stages of the viral infection, in order to be used as target of prophylactic actions. A fish cell line (SSN-1) was used as model to tentatively develop in vitro studies. Only defective particles were produced confirming the high specificity to crustacea of the infection with crustacean virus. Electron microscopy showed structural similarities between the WSSV and B, B2 and Baculo-B viruses of crabs. This suggests B2 may belong also to the family Nimaviridae, genus Whispovirus. This comparison with B2 virus gives the possibility to understand the role played by the tail-like extension of these viruses in the infectious process by attachment to the plasmic membrane at the beginning of the infection in its specific host. Les crevettes de grande taille de la famille des Penaeidae constituent le premier produit aquacole en valeur commerciale à l'échelle planétaire. Elles sont produites à 99% dans les pays en voie de développement de la ceinture sub-équatoriale. Parmi les causes limitant leur production, le White Spot Syndrome Virus est l'agent pathogène ayant provoqué le plus des pertes dans le monde. Notre étude a été orientée vers la reconnaissance des premiers stades de l'infection, susceptibles d'être la cible d'une action prophylactique. Nous avons testé l'utilisation d'une lignée cellulaire de poisson SSN-1 afin d'étudier les possibilités d'un développement in vitro. Seules des particules défectives ont été produites confirmant la haute spécificité des infections à virus de crustacés. Les recherches en microscopie électronique ont montré une similarité structurale du WSSV avec les virus (B, B2 et Baculo-B) des crabes. Ceci suggère que ces agents (B2 et WSSV) seraient tout deux de la famille des Nimaviridae et du genre Whispovirus. La comparaison avec le virus B2 permet de comprendre le rôle clef joué par la partie caudale de ces virus dans l'infection par son attachement à la membrane plasmique lors de l'infection chez son hôte spécifique.
... Given the economic significance of WTD, there is an urgent need for easy and reliable detection methods. Different diagnostic methods are available to detect MrNV/XSV in prawn and among these methods, RT-PCR is the most sensitive (Romestand & Bonami 2003;Sri Widada et al. 2003;Sahul Hameed et al. 2004a;Sri Widada, Richard, Cambournac, Shi, Qian & Bonami 2004;Yoganandhan, Sri Widada, Bonami & Sahul Hameed 2005). As both viruses infect M. rosenbergii by vertical and horizontal transmission, it is desirable to screen for them in broodstock before spawning and in seed before stocking ponds. ...
White tail disease (WTD) is a serious problem in hatcheries and nursery ponds of Macrobrachium rosenbergii in India. Experiments were carried out to determine the possibility of vertical transmission of M. rosenbergii nodavirus (MrNV) and extra small virus (XSV) in M. rosenbergii and Artemia. Prawn broodstock inoculated with MrNV and XSV by oral or immersion challenge survived without any clinical signs of WTD. The brooders spawned 5-7 days after inoculation and the eggs hatched. The survival rate of larvae gradually decreased, and 100% mortality was observed at the post-larvae (PL) stage. Whitish muscle, the typical sign of WTD, was seen in advanced larval developmental stages. The ovarian tissue and fertilized eggs were found to be positive for MrNV/XSV by reverse transcriptase-polymerase chain reaction (RT-PCR) whereas the larval stages showed positive by RT nested PCR (nRT-PCR). In Artemia, reproductive cysts and nauplii derived from challenged brooders were normal and survival rates were within the expected range for normal rearing conditions. The reproductive cysts were found to be positive for MrNV/XSV by RT-PCR whereas the nauplii showed MrNV/XSV-positive by nRT-PCR. The PL of M. rosenbergii fed nauplii derived from challenged Artemia brooders died at 9 days post-inoculum with clinical signs of WTD.
... Dot blot hybridization and RT-PCR have also been developed for detection of XSV (Sri Widada, Richard, Shi, Qian & Bonami 2004 ). A singletube , one-step multiplex RT-PCR has been developed for detecting MrNV and XSV (Yoganandhan, Sri Widada, Bonami & Sahul Hameed 2005). Although the relationships between MrNV and XSV virus remains unknown, it is hypothesized that XSV constitutes a new species of satellite virus (). ...
Post-larvae of Macrobrachium rosenbergii infected with white tail disease (WTD) have been reported in Taiwan. The causative agents have been identified as M. rosenbergii nodavirus (MrNV) associated with extra small virus (XSV). The present study is the first report confirming the presence of XSV virus in M. rosenbergii displaying WTD symptoms in Taiwan by reverse transcription polymerase chain reaction (RT-PCR). A 772 bp amplified product was obtained by RT-PCR, cloned and sequenced. The nucleotide sequence analysis of the 772 bp DNA fragment revealed 98% and 97% identity with XSV isolated from China and India, respectively. Comparison of the deduced amino acid sequences of the XSV partial genome showed strong homology (99% and 97%) with isolates from China and India. Phylogenetic analysis revealed the XSV-Taiwan isolate was more closely related to the Chinese rather than the Indian isolates. The results demonstrated the presence of XSV virus co-infection in M. rosenbergii cultured in Taiwan suffering from WTD.
... Its genome is formed by two pieces of ss RNA of 2.90 and 1.26 kb, respectively. (Sri Widada et al. 2003;Yoganandhan, Sri Widada, Bonami & Sahul Hameed 2005). Satellite virus-like particles (named XSV) are 15 nm in diameter with an ss RNA genome of 0.8-0.9 kb coding only for its capsid protein but lacking the polymerase gene (RdRp); thus, XSV is unable to replicate alone. ...
This study evaluated the possible use of the fish SSN-1 cell line to investigate the development of Macrobrachium rosenbergii nodavirus (MrNV). Cells were incubated with viral particles and cytopathic effects were observed. De novo synthesis of viral capsid proteins was shown by immuno-fluorescence labelling and a sandwich ELISA test. Viral genomic replication was demonstrated by RT-PCR using primers specific to RNA-1 as well as by quantitative RT-PCR (RT-qPCR). Using electron microscopy, only a few empty particles were observed and attempts to isolate complete infectious particles or to re-infect healthy cells (second passage) were unsuccessful. As complete viral particles were rarely observed, it appeared that defaults in MrNV virogenesis might arise resulting in the formation of scarce and non-infectious particles. SSN-1 cells were found to be partially permissive to MrNV infection that induced cell lysis, but key elements for viral infection were lacking such as regulatory factors for gene replication or post-translational modifications.
White spot syndrome virus (WSSV), Enterocytozoon hepatopenaei (EHP), and Acute hepatopancreatic necrosis disease (AHPND) are the major threats to the whiteleg shrimp, Litopenaeus vannamei . This study was conducted to develop multiplex polymerase chain reaction (M-PCR) for the detection of shrimp pathogens. This study was conducted between January 2020 to July 2022. Total of 328 samples were collected from shrimp farms of Karnataka and they were screened for WSSV, EHP, and AHPND by M-PCR. Several parameters were optimized for the standardization of M-PCR. The specificity and sensitivity of the reaction were evaluated. The results showed that this technique can detect WSSV, AHPND, and EHP in a single reaction with high specificity. Sensitivity result showed it can detect WSSV (0.3 pg/µl), AHPND (0.1 pg/µl), and EHP (0.2 pg/µl). Out of 328 samples, 72 (21.9%) samples were found to be positive for EHP, 16 (4.8%) samples were found to have been infected by WSSV and 5 (1.5%) samples were found to have multiple infections with WSSV, and EHP. In consideration of the specificity and sensitivity of this technique, we conclude that M-PCR could be used instead of a conventional PCR assay targeting individual genes for the rapid detection of WSSV, AHPND, and EHP simultaneously.
This study detected two potential pathogens, Vibro parahaemolyticus, which causes acute hepatopancreatic necrosis disease (AHPND), and white spot syndrome virus (WSSV), in fishing bait in South Korea. However, their infectious nature was not confirmed, possibly due to the degradation caused by freezing/thawing or prolonged storage under frozen conditions. While infectivity was not confirmed in this study, there is still a significant risk of exposure to these aquatic products. Furthermore, fishing bait and feed should be handled with caution as they are directly exposed to water, increasing the risk of disease transmission. In Australia, cases of WSSV infection caused by imported shrimp intended for human consumption have occurred, highlighting the need for preventive measures. While freezing/thawing is a method for inactivating pathogens, there are still regulatory and realistic issues to be addressed.
The giant river prawn, Macrobrachium rosenbergii, is a major focus of aquaculture in tropical and sub‐tropical regions around the globe. Over the last 30 years, culture of M. rosenbergii has increased exponentially as demand has risen both for domestic consumption and for international export trade. As with many aquaculture species increases in production have been accompanied by the emergence of diseases affecting yield, profit and trading potential. Disease‐causing agents include pathogens infecting other crustaceans, such as Decapod Iridescent Virus (DIV1), as well as pathogens only known from M. rosenbergii such as White Tail Disease caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV). Here, we review the pathogenic agents associated with the culture of M. rosenbergii since commercial culture began in earnest during the 1970s. Particular emphasis is given to pathogens first identified in other aquaculture host species, but which have subsequently been shown to infect and cause disease in M. rosenbergii. As polyculture of M. rosenbergii with other aquaculture species is common practice, including culture with other decapods, crabs and fish, increased pathogen transfer among these farmed species may occur as M. rosenbergii aquaculture increases in the future.
White tail disease (WTD) is a serious viral disease in the hatcheries and nursery ponds of Macrobrachium rosenbergii in many parts of the world. The virus which causes WTD was named Macrobrachium rosenbergii nodavirus (MrNV). WTD mostly affects the young prawn. It may cause severe mortalities in postlarvae but no mortality in the infected adult prawn. Besides the freshwater prawn, WTD could affect and cause high mortality in some penaeid species. Penaeus (Litopenaeus) vannamei has been reported to be susceptible to MrNV. Experimental transmission of WTD in adult Penaeus monodon, Penaeus indicus, and P. japonicus showed no mortalities. This chapter will discuss important aspects of the pathophysiology of WTD, supported by a review of the diagnosis and control of the disease.
Macrobrachium rosenbergii nodavirus (MrNV) affects the larval, post-larval, and juvenile stages of M. rosenbergii, the giant freshwater prawn, causing white tail disease (WTD). With its high mortality, WTD is a severe threat to shrimp and prawn farming. We describe the development and optimization of an antibody-based lateral flow assay (LFA) for the early detection of MrNV in the post-larval (PL) stage of M. rosenbergii. The LFA parameters (viz., the detergent concentration, GNPs-antibody conjugate, antibody concentration applied to the test line and membrane porosity) were optimized using the design of experiment (L9 orthogonal array). Under optimized conditions, MrNV could be detected within 20 min with high specificity, reproducibility, and sensitivity (LOD = 104 particles/ng of total RNA). In virus challenge experiments, MrNV could be detected on the seventh day of infection in the PL stage. LFA was validated using infected PL samples collected from the field (hatcheries and nurseries) (n = 80) in conjunction with ‘gold standard’ qRT-PCR test. High sensitivity (100%) and specificity (90%) of LFA, with a Cohen's kappa coefficient of 0.936, suggested ‘good agreement’ between the developed LFA and qRT-PCR. The developed LFA has an immense potential of averting losses by rapid detection of MrNV at PL stage in M. rosenbergii.
To date, only three nodaviral diseases have been reported in crustaceans. White tail disease (WTD), caused by Macrobrachium rosenbergii nodavirus (MrNV), was reported first and identified after clinical signs were observed in the postlarval of M. rosenbergii. The second nodaviral disease was caused by Penaeus vannamei nodavirus (PvNV), and was identified in 2004. Since 2009, a new nodavirus, covert mortality nodavirus (CMNV), has been reported as causing covert mortality disease in China. The gross signs of shrimp infected by MrNV, PvNV or CMNV are indistinguishable. However, these three nodaviruses differ in virulence. Viral genome sequences data suggested that MrNV, PvNV and CMNV are affiliated with the Nodaviridae family. Specific and sensitive detection methods are available for MrNV, PvNV and CMNV identification. Several MrNV prevention and control methods have been investigated in laboratory settings; however, no such trials for PvNV or CMNV have been performed in this regard.
Nervous necrosis virus (NNV) infected larvae and juveniles of more than 50 fish species, resulting in mortality rates of greater than 95%. However, there is no efficient method to control NNV infections. Aptamers generated by selective evolution of ligands by exponential enrichment (SELEX) are short, single-stranded nucleic acid oligomers. They display a high degree of affinity and specificity for many targets, such as viruses and viral proteins. In this study, three novel DNA aptamers (A5, A10, and B11) that specifically target the coat protein (CP) of grouper nervous necrosis virus (GNNV) were selected using SELEX. Secondary structures and minimum free energy (ΔG) predictions indicated that these aptamers could form stable, secondary stem-loop structures. Electrophoretic mobility shift assays, enzyme-linked immunosorbent assays, Kd measurements, the co-localization of tetramethylrhodamine (TAMRA) labeled-aptamers with the CP and flow cytometry analysis revealed that these aptamers could specifically bind the CP with high (nanomolar) affinities. In addition, competition analysis suggested the aptamers shared some common CP binding sites with the anti-CP antibody. Moreover, all three aptamers did not show any cytotoxic effects in vitro or in vivo, and anti-viral analysis indicated the selected aptamers could inhibit NNV infection in vitro and in vivo. Compared with controls, mortality of GNNV-infected fish decreased by 40% and 80% after 10 days infection, when the GNNV was pre-incubated with the 1000 nM A10 and B11, respectively. TAMRA-labeled aptamers could bind to NNV virions and directly enter NNV-infected cells, suggesting they could be used as tracers to study the mechanism of viral infection, as well as for targeted therapy. This is the first time that aptamers targeting a viral protein of marine fish have been generated and characterized. These aptamers hold promise as diagnostic, therapeutic, and targeted drug delivery agents for controlling NNV infections.
A total number of 500 specimens of Macrobrachium rosenbergii were collected during (2011) from Alexandria (Maruit). Each five specimens were pooled for extraction of RNA and carrying out of RTPCR analysis. These specimens were transferred to the laboratory of Central Laboratory for Aquaculture Research (CLAR), Abbassa, Sharkia, Egypt for evaluation of different reverse transcriptase polymerse chain reaction (rt-PCR) assays for diagnosis of Macrobrachium rosenbergii nodavirus (MrNV), and extra small virus (XSV). Multiplex reverse transcriptase Polymerase chain reaction (MRT-PCR) assay for simultaneous detection of MrNV and XSV were classified into one-step MRTPCR and two steps MRTPCR. Results of one-step multiplex (MRTPCR) using (BioRT One step RT-PCR kit) and primer for MrNV virus (product size 681 bp) were negative. Also, the results of One-step multiplex (MRTPCR) using primer for XSV (product size 500 bp) were negative. In addition, results of two steps multiplex MRTPCR using (GoScript™ Reverse Transcription System kits), primer for MrNV (product size 681 bp) and primer Oligo(dT)15 for obtaining first CDNA were negative. Where, percentage of infections of two steps multiplex RTPCR using primer for XSV (product size 500 bp) and primer Oligo(dT)15 for obtaining first CDNA were (4%). Percentage of infections of nested (nRTPCR) for detection of Mrnv virus using (AccessQuick™ RTPCR kit), primer amplification (product size 205 bp) were 9%. Nested (nRTPCR) for detection of XSV virus using (AccessQuick™ RT-PCR kit), primer amplification (product size 236 bp) were 7%.
The giant freshwater prawn, Macrobrachium rosenbergii de Man or scampi is an economically important farmed crustacean species all over the world. India contributes ~ 20% to the world's scampi aquaculture production, being ranked second in the world In recent years, the rapid expansion and intensification of culture practices have brought several diseases of infectious and noninfectious aetiologies. The increased globalization of trade and transboundry movement of broodstock and postlarvae, the unanticipated interactions between cultured and wild populations, poor biosecurity measures, lack of awareness on emerging pathogens, climate change, misuse of drugs and antibiotics and other human-mediated movement of aquaculture produce and products are playing key role in occurrence, spread and outbreaks of diseases. The main causative agents of the infectious diseases are viruses, bacteria, rickettsia, fungi and protozoa. In past few years, nodavirus is causing a devastating production loss to scampi industry in many countries including India. So disease control is becoming a priority. The defence mechanism of giant freshwater prawn, M rosenbergu de Man. is poorly understood The knowledge of the functioning of its defence system is of extreme importance. The stimulation of this system is considered as a potential intervention strategy in scampi culture to overcome the infectious diseases. This review focuses on the recent information of major diseases of giant freshwater prawn, M rosenbergu de Man. and the related defence mechanism which may be of help for sustainable development of fast-growing scampi industry.
Genetic parameters and response to selection were estimated for harvest body weight of the giant freshwater prawn Macrobrachium rosenbergii using a fully pedigreed synthetic population from three introduced strains. The data included 65,917 progeny that were from 331 sires and 544 dams in five generations with a nested mating structure. Harvest body weight was transformed by square root for analysis. Variance components and genetic parameters were estimated using an animal model and the restricted maximum likelihood method. The estimated breeding values of all animals over five generations were calculated using best linear unbiased prediction. Within generations, the heritability estimates for harvest body weight in each generation ranged from low to moderate (0.055 ± 0.012 to 0.223 ± 0.045) and were significantly different from zero (P < 0.05). Only the common environmental effect of the three generations from G1 to G3 could be estimated, which was found to vary from 0.024 ± 0.012 to 0.032 ± 0.014. Across generations, the heritability and common environmental effect estimates were low (0.056 ± 0.014 and 0.039 ± 0.005, respectively) and significantly different from zero (P < 0.05). Heritability of harvest body weight in females was significantly higher than that in males across populations. However, the genetic correlation of harvest body weight between sexes across population was close to unity (0.942 ± 0.070), indicating that body weight in male and female shrimp is most likely controlled by the same genes. The response to selection in harvest body weight was estimated by two methods (the realised and predicted responses). The realised response was estimated from the difference in the least squares means of body weight for the selection and control populations, while the predicted response was obtained from the difference in the mean breeding values between generations. The back-transformed realised response was 26.22%, while the predicted responses estimated using two set of genetic parameters obtained from within- and across-generations datasets were 18.06% and 12.38% in actual units, respectively, after performing four selections. The results are discussed in relation to selection work with other farmed shrimps, and solutions for increasing and disseminating genetic gain are outlined.
Rapid, sensitive, and automatic detection platforms are among the major approaches of controlling viral diseases in aquaculture. An efficient detection platform permits the monitoring of pathogen spread and helps to enhance the economic benefits of commercial aquaculture. Nervous necrosis virus (NNV), the cause of viral encephalopathy and retinopathy, is among the most devastating aquaculture viruses that infect marine fish species worldwide. In the present study, a highly sensitive magnetoreduction assay was developed for detecting target biomolecules with a primary focus on NNV antigens. A standard curve of the different NNV concentrations that were isolated from infected Malabar grouper (Epinephelus malabaricus) was established before experiments were conducted. The test solution was prepared by homogeneous dispersion of magnetic nanoparticles coated with rabbit anti-NNV antibody. The magnetic nanoparticles in the solution were oscillated by magnetic interaction with multiple externally applied, alternating current magnetic fields. The assay's limit of detection was approximately 2 × 10(1) TCID(50)/ml for NNV. Moreover, the immunomagnetic reduction readings for other aquatic viruses (i.e., 1 × 10(7) TCID(50)/ml for Infectious pancreatic necrosis virus and 1 × 10(6.5) TCID(50)/ml for grouper iridovirus) were below the background noise in the NNV solution, demonstrating the specificity of the new detection platform.
The capsid protein (CP) gene of extra small virus (XSV) expressed in Escherichia coli as a 42 kDa glutathione S-transferase (GST)-fusion protein (GST-XCP) or a 20 kDa His6-fusion protein (His6-XCP) were purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), combined, and used to immunize Swiss mice to produce monoclonal antibodies (MAbs). Using dot blot, Western blot, and immunohistochemistry (IHC) methods, 4 MAbs specific to the XSV CP detected XSV in the freshwater prawn Macrobrachium rosenbergii without cross-reaction to host proteins or to proteins of Macrobrachium rosenbergii nodavirus (MrNV) or 5 of the most pathogenic viruses of penaeid shrimp. In dot blots, the combined MAbs could detect down to ~10 to 20 fmol µl-1 of purified GST-XCP protein, which was somewhat more sensitive compared to any single MAb. Used in conjunction with an MrNV-specific MAb, white tail disease (WTD) was diagnosed more effectively. However, the sensitivity at which the combined 4 MAbs detected XSV CP was 1000-fold lower than XSV RNA detected by RT-PCR. IHC analysis of M. rosenbergii tissue sections using the MAbs showed XSV infection to co-localize at variable loads with MrNV infection in heart and muscle cells as well as cells of connective tissues in the hepatopancreas. Since XSV histopathology remained prominent in tissues of some prawns in which MAb reactivity for MrNV was low compared to MAb reactivity for XSV, XSV might play some role in WTD severity.
Intensive prawn farming is characterised by the confinement and husbandry of the population in artificially constructed production systems, such as hatchery tanks and grow-out ponds. Various biotic or abiotic factors within intensive systems challenge the health of cultured populations and failure to control them usually leads to feeble or even disastrous production results. Some suggest (see Bédier et al. 1998) that infectious diseases, mainly viruses, have been responsible for the collapse of many marine shrimp farming ventures in the past few years. Important diseases have also been associated with freshwater prawn (Macrobrachium rosenbergii) culture which could seriously affect commercial production but virus diseases that would threaten the industry on a large scale have not been reported. Recently, in a paper describing the culture of M. rosenbergii and M. nipponense in China,Wang & Qianhong (1999) reported that, though they are not as serious as those for penaeids, diseases have already jeopardised the development of freshwater prawn culture. These authors particularly noted that mortalities of broodstock occurred during the overwintering period and that 'white shrimp disease' occurred during the grow-out period.Apart from the obvious negative impact that diseases may pose to prawn production, some diseases compromise marketability of the final product by rendering poor flesh quality (muscular necrosis) or undesirable aesthetic changes (black spot or heavy fouling by epibionts). Though the body of literature concerning prawn disease contains many excellent scientific works, the composite is small in comparison to that available for many other aquaculture animals. This may be partly due to the growth in the importance of penaeid culture during the 1980s and 1990s, a circumstance that attracted the attention of most of the active crustacean health specialists. Before describing the specific diseases and other problems that have been found to affect freshwater prawns (section 15.5), relevant information on health and defence mechanisms (section 15.1), diagnosis (section 15.2), prophylaxis (section 15.3) and therapeutics (section 15.4) has been reviewed in this chapter.
White tail disease (WTD) of the freshwater prawn Macrobrachium rosenbergii has recently been the cause of high mortalities in Thai prawn farms. The causative agents of this disease in other countries are M. rosenbergii nodavirus (MrNV) and extra small virus (XSV), which are usually detected using reverse transcriptase-polymerase chain reaction (RT-PCR) protocols. Using RT-PCR, most Thai post-larvae (PL) samples showing gross signs of WTD tested positive for MrNV but only a few were positive for XSV. In contrast, all tested brooder samples were positive for both MrNV and XSV. The possibility that brooders infected with MrNV and XSV could transmit the viruses to larvae and PL should be examined. Cloning, sequencing and comparison of deduced amino acid sequences of RT-PCR amplicons of WTD samples from Thailand with those of MrNV and XSV previously reported from the French West Indies and China revealed that the MrNV were closely related but not identical while those from XSV were identical. This is the first report of MrNV and XSV from Thailand.
Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) were purified from diseased freshwater prawns M. rosenbergii and used to infect healthy post-larvae (PL) by an immersion method. Three groups of prawns were challenged with various combined doses of MrNV and XSV. Signs of white-tail disease (WTD) were observed in Groups 1 and 2, which had been challenged with combinations containing relatively high proportions of MrNV and low proportions of XSV. By contrast there was little sign of WTD in Group 3, which had been challenged with a higher proportion of XSV than MrNV. A 2-step Taqman real-time RT-PCR was developed and applied to quantify viral copy numbers in each challenged PL. Results showed that genomic copies of both viruses were much higher in Groups 1 and 2 than they were in Group 3, indicating that MrNV plays a key role in WTD of M. rosenbergii. The linear correlation between MrNV and XSV genome copies in infected prawns demonstrated that XSV is a satellite virus, dependent on MrNV, but its role in pathogenicity of WTD remains unclear.
White tail disease (WTD) is a serious problem in Macrobrachium rosenbergii hatcheries and nursery ponds in Asia. The causative agents have been identified as M. rosenbergii nodavirus (MrNV) and its associated extra small virus. This is the first report demonstrating MrNV virus in M. rosenbergii displaying WTD signs in Taiwan by reverse transcriptase-polymerase chain reaction (RT-PCR). Amplified fragments of 850 and 425 bp for RNA-1 and RNA-2 of MrNV, respectively, were obtained by RT-PCR. RT-PCR products of about 850 and 1121 bp for RNA-1 and RNA-2 of MrNV were also obtained using different primer pairs. The amplicons were individually cloned into pGEM-T vector and sequenced. Using this recombinant plasmid of MrNV RNA-2 as DNA template, the non-radioactive DNA probes were prepared by PCR amplification with DIG-11-dUTP. The probes were used to successfully detect MrNV infection in the striated muscle tissues of WTD-diseased prawns using in situ hybridization. The 1121 bp genomic fragment of RNA-2 of MrNV consisted of a unique open reading frame with 1116 nucleotides, and it encoded a structural protein with 371 amino acids. The nucleotide sequence of the partial genome of MrNV RNA-2 revealed a 97% identity with an Indian isolate. A phylogenetic tree constructed using the nucleotide sequence of the viral capsid gene from insect and fish nodaviruses revealed that the MrNV Taiwan isolate could be interpreted as a new genus within the family Nodaviridae. However, its position showed more affinity with Alphanodavirus than with Betanodavirus. The study confirmed the presence of MrNV infection in freshwater prawns cultured in Taiwan suffering from WTD.
Penaeid acute viremia (PAV) caused by penaeid rod-shaped DNA virus (PRDV: one of white spot baculovirus complex) is the most serious disease affecting kuruma prawn (Penaeus japonicus) culture in Japan. In this study an experimental challenge was carried out in different larval (nauplius, zoea and mysis) and postlarval (PL 1, 6, 9 and 11-12) stages of kuruma prawn in order to elucidate the difference in susceptibility to PRDV among different developmental stages of the host. The PRDV challenge was done by immersion in all the tested groups. No infections were recorded in the larval stages and PL1. The PL6 first showed mortality due to PAV on 10th day post-inoculation. The PL9 exhibited the onset of PAV on 5th day post-inoculation. In PL11-12, experimental PAV was produced more rapidly and resulted in higher mortality (78.6%). These results demonstrate that the PRDV-susceptibility of P. japonicus increases with the progress in developmental stage until PL12 and that PRDV may not display its pathogenicity in the larval and early postlarval stages younger than PL6.
Penaeid acute viremia (PAV) caused by penaeid rod-shaped DNA virus (PRDV: one of white spot baculovirus complex) is the most serious disease affecting kuruma prawn (Penaeus japonicus) culture in Japan. In this study an experimental challenge was carried out in different larval (nauplius, zoea and mysis) and postlarval (PL 1, 6, 9 and 11-12) stages of kuruma prawn in order to elucidate the difference in susceptibility to PRDV among different developmental stages of the host. The PRDV challenge was done by immersion in all the tested groups. No infections were recorded in the larval stages and PL1. The PL6 first showed mortality due to PAV on 10th day post-inoculation. The PL9 exhibited the onset of PAV on 5th day post-inoculation. In PL11-12, experimental PAV was produced more rapidly and resulted in higher mortality (78.6%). These results demonstrate that the PRDV-susceptibility of P. japonicus increases with the progress in developmental stage until PL12 and that PRDV may not display its pathogenicity in the larval and early postlarval stages younger than PL6.
In the course of experimental infection of Penaeus monodon with yellow-head virus (YHV) for virus isolation and purification, 1 batch of prawns yielded hemolymph fractions dominated by a previously undescribed non-occluded baculovirus rather than YHV. Injection of test shrimp with a semi-purified preparation of this virus gave rapid mortality, and examination with the transmission electron microscope revealed a dual infection where cells containing the new virus dominated, but some cells containing YHV could also be seen. The tissues infected by the 2 viruses were similar. However, in contrast to YHV, the new virus was assembled completely in the nucleus and in the absence of occluding protein (polyhedrin). By normal histology, the most characteristic feature of infection was eosinophilic Cowdry A-type inclusions in hypertrophied nuclei with marginated chromatin, especially in epithelial cells of the stomach. These intranuclear inclusions became lightly basophilic in late stages of infection. In the epithelial cells of the gills, ultrastructural pathology included nuclear hypertrophy and cytoplasmic disintegration leading to large voids at lysed cell sites. By negative staining, completely assembled, enveloped virions were ellipsoid to obovate with a distinctive multifibrillar appendage and they measured 276 x 121 nm (excluding the appendage). Enveloped and unenveloped nucleocapsids were significantly different in size, indicating possible shortening and thickening of the viral core and nucleocapsid during viral assembly. isolation and purification of the nucleic acid from the new virus yielded double-stranded DNA of approximately 168 kilo base pairs. This DNA did not cross-hybridize with DNA fragments isolated from YHV-infected shrimp or from monodon baculovirus (MBV). The features placed this virus in the family Baculoviridae, subfamily Nudibaculovirinae as PmNOBII, but for convenience we have named it informally as Systemic Ectodermal and Mesodermal Baculovirus (SEMBV).
Τhe giant freshwater prawn, Macrobrachium rosenbergii (de Man), has been one of the major aquacultural products of Taiwan for the past decade. The annual production of cultured freshwater prawns increased from 1477 MT in 1982 to 16 196 MT in 1991, and in recent years, these animals have become the second most important cultured crustacean crop after the marine prawn, Penaeus monodon (Fabricius). Since 1992, the postlarvae of freshwater prawns in southern Taiwan have been affected by an epizootic disease similar to idiopathic muscle necrosis (IMN) syndrome (Akiyama, Brock & Haley 1982| Anderson, Nash & Shariff 1990b). The affected prawns exhibit white opaque areas in abdominal segments, commonly accompanied by progressive weakening of their feeding and swimming ability. The histopathological changes are similar to those of IMN syndrome, as described by previous studies (Nash, Chinabut & Limsuwan 1987). Unlike IMN, a cytoplasmic inclusion body has also been detected in the necrotic muscle of diseased prawns. Electron microscopy revealed icosahedral virus particles in the cytoplasm of necrotic cells as well as aggregations of viral particles in the inclusion body. The virus is temporarily named Macrobrachium muscle virus (MMV) until its taxonomical position is ascertained by analysing the structure of the genomic DNA. The present paper discusses the first histopathological and ultrastructural observation of MMV infection in cultured freshwater prawn postlarvae from Taiwan.
The availability of specific and reliable detection methods is essential for monitoring the health status of farmed species, particularly for viral diseases. Extra small virus (XSV), a virus-like particle, is associated with Macrobrachium rosenbergii Noda virus (MrNV) in white tail disease (WTD) of M. rosenbergii. We developed 2 genome-based detection methods for the identification of XSV, namely dot-blot hybridization and a single-step RT-PCR. Detection limits were established and are ca. 2.5 pg and 5 fg of viral RNA for dot-blot hybridization and RT-PCR, respectively. Application of the methods to field samples indicated that some animals positively diagnosed with MrNV did not contain XSV, at least within the detection limit of the methodology. This raises the question of the actual role of XSV and its interactions with MrNV in WTD of M. rosenbergii.
White tail disease (WTD) was found to be a serious problem in hatcheries and nursery ponds of Macrobrachium rosenbergii in India. The causative organisms have been identified as M. rosenbergii nodavirus (MrNV) and its associated extra small virus (XSV). Experimentally transmitted to healthy animals, they caused 100% mortality in post-larvae but failed to cause mortality in adult prawns. The RT-PCR assay revealed the presence of both viruses in moribund post-larvae and in gill tissue, head muscle, stomach, intestine, heart, hemolymph, pleopods, ovaries and tail muscle, but not in eyestalks or the hepatopancreas of experimentally infected adult prawns. The presence of these viruses in ovarian tissue indicates the possibility of vertical transmission. Pleopods have been found to be a suitable organ for detecting these viruses in brooders using the RT-PCR technique.
During mortality outbreaks in hatchery-reared Macrobrachium rosenbergii postlarvae (PL) in Guadeloupe Island (French West Indies) during 1997, an associated viral disease was discovered and the agent was subsequently isolated. The clinical signs presented by severely affected PL consisted essentially of an opaque whitish appearance of the abdomen. Histopathological changes in affected PL were characterized predominantly by pale to darkly basophilic, often reticulated, cytoplasmic inclusions in the connective tissue cells of most organs and tissues. The isolated virus was approximately 30 nm in diameter as observed with an electron microscope by negative staining. By its location, structure and size it could be related to different families of the small RNA cytoplasmic viruses such as the Picornaviridae or the Nodaviridae. Its characterization is in progress.
A new disease similar to whitish tail disease (WTD) was observed in larvae and post-larvae of Macrobrachium rosenbergii in hatcheries and nursery ponds located at Nellore, Vijayawada and Chennai, India. Reverse transcriptase polymerase chain reaction (RT-PCR) assay on these samples revealed the presence of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) in infected larvae and post-larvae, as reported in China. Published primers and self-designed primers specific for MrNV were tested and compared for sensitivity with MrNV samples collected in India. Our designed primer sets 4 and 2 gave amplicons of 425 and 590 bp, respectively, with improved sensitivity to the level of 0.25 fg of total RNA when compared to 25 fg of total RNA for the previously published method. XSV detection was based on a recently published RT-PCR protocol. This is the first report on the occurrence of MrNV and XSV in WTD of freshwater prawn from India.
The most important microbial diseases encountered in the rapidly developing mariculture industry of Japan are described. Vibriosis, pseudotuberculosis, streptococcicosis, nocardiosis and bacterial kidney disease (BKD) were the most important bacterial infections, resulting insignificant losses in mariculture fish production. Viral infections with epizootic outbreaks were observed with yellowtail fry and Japanese flounder. In kuruma shrimp culture, problems were caused by vibriosis and a Baculovirus sp. The annual damage and losses due to diseases in mariculture in Japan in 1984 are tabulated. An overview is given of the drugs allowed and used for treatment of diseases of marine fish in Japan.
White spot syndrome virus (WSSV), the causative agent of white spot syndrome in shrimp, has a wide host range which extends to crabs, copepods and other arthropods. In this study, benthic larvae of the mud crab Scylla serrata were captured from Taiwan's coastal waters and screened for the presence of WSSV by polymerase chain reaction (PCR) and in situ hybridization. WSSV was detected in around 60% of the larvae, and this prevalence rate remained fairly constant when the captured larvae were subsequently maintained in an aerated system in the laboratory. WSSV-free larvae obtained from a hatchery were challenged by immersion in a WSSV inoculum. Fifteen days after challenge, cumulative mortality in the experimental group reached 43% compared to 20% in the control group. PCR detection of WSSV in both moribund and surviving specimens clearly implicated the virus as the cause of death in most cases. Histological and in situ hybridization data confirmed that WSSV tissue tropism in Scylla serrata crab larvae is similar to that found in shrimp.
A sandwich enzyme-linked immunosorbent assay (S-ELISA) was developed to improve diagnosis of white tail disease of the giant freshwater prawn, Macrobrachium rosenbergii, caused by the nodavirus, MrNV. Polyclonal antibodies were produced by immunization of Balb/C mice using a purified suspension of the virus and IgG anti-MrNV were purified from ascitic fluid. A sandwich method was successfully developed, coating first with unlabelled antibody and detecting trapped antigens with a second biotinylated antibody. Reaction was demonstrated using an avidin-peroxidase conjugate. Tissue extracts from M. rosenbergii infected with MrNV or purified viral extracts (control) were successfully identified in an individual ELISA, thus confirming the validity of the method. This S-ELISA should be the technique of choice for epidemiological studies of this disease and is a rapid and inexpensive assay with high specificity and sensitivity.
A disease of Macrobrachium rosenbergii, the giant freshwater prawn, farmed in China was recently recorded in Zhejiang, Jiangsu, Shanghai, Guangxi and Guangdong provinces. The clinical sign of the disease, which develops in post-larvae (PL), is a whitish appearance of the muscles, particularly noticeable in the abdomen. Mortalities may reach 100% in some hatcheries. Investigations by transmission electron microscopy after negative staining of diseased PL homogenates showed the presence of two types of viral particles: one, unenveloped, icosahedral in shape, 26-27 nm in diameter, the second, much smaller, about 14-16 nm in diameter, designated extra small virus particle (XSV). The large virus has a genome with two pieces of ssRNA (RNA-1 and RNA-2), of 3 and 1.2 kb, respectively. Hybridization tests confirmed that this large virus is closely related to M. rosenbergii nodavirus (MrNV) which was isolated from diseased prawns in a hatchery in the French West Indies. Its very small size and hypothesized biochemical and biological characteristics suggest XSV is a new type of crustacean virus. As XSV has always been found associated with the larger virus (nodavirus) and is located in muscle and connective cells of diseased animals, it could be an autonomous virus, a helper-type virus or a satellite-like virus.
The availability of specific and rapid detection methods is essential for monitoring the health status of farmed species, particularly in viral diseases as in this case early diagnosis is a critical factor in containing disease outbreaks. Three complementary genome-based methods were developed for the detection of Macrobrachium rosenbergii nodavirus (MrNV), i.e. dot-blot hybridization, in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR). Detection limits were established for dot-blot hybridization and RT-PCR and are c. 7 fg and 8 pg of viral RNA, respectively. In situ hybridization indicated that infection was confined to the striated muscle tissue. As a result of its sensitivity, RT-PCR can be used for in-depth investigations to examine the extent of the viral infection and establish the onset of infection in hatcheries. The application of RT-PCR on samples collected from prawn farms in China showed the possible use of this method in routine health monitoring.