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Experimental transmission of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) in three species of marine shrimp (Penaeus indicus, Penaeus japonicus and Penaeus monodon)
Aquaculture 257 (2006) 136 – 141 www.elsevier.com/locate/aqua-online
Experimental transmission of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) in three species of marine shrimp (Penaeus indicus, Penaeus japonicus and Penaeus monodon) R. Sudhakaran a , S. Syed Musthaq a , P. Haribabu b , S.C. Mukherjee b , C. Gopal c , A.S. Sahul Hameed a,⁎ a
Department of Zoology, C.Abdul Hakeem College, Melvisharam-632 509, Vellore Dist., Tamil Nadu, India b Central Institute of Fisheries Education, Fisheries University Road, Versova, Mumbai-400 061, India c Central Institute of Brackishwater Aquaclture, Santhom High Road, R.A. Puram, Chennai-600 028, India Received 27 September 2005; received in revised form 21 February 2006; accepted 22 February 2006
Abstract The susceptibility of three species of marine shrimp (Penaeus indicus, Penaeus japonicus and Penaeus monodon) to Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) was tested by oral route and intramuscular injection. The results revealed that these marine shrimp were not susceptible to these viruses which failed to produce mortality in shrimp. RTPCR analysis revealed the presence of MrNV and XSV in different organs such as gill, abdominal muscle, stomach, intestine and hemolymph of three species of shrimp injected with viruses. These viruses were also found in different tissues of shrimp fed with WTD-infected prawn meat, but not in control groups fed with uninfected meat. The reinoculation studies using the inoculum of MrNV and XSV prepared from marine shrimp caused 100% mortality in the post-larvae of freshwater prawn and the moribund post-larvae showed positive for these viruses by RT-PCR. The results of present study indicate the possibility of marine shrimp acting as reservoir for MrNV and XSV and maintaining their virulence in tissue system of marine shrimp. © 2006 Elsevier B.V. All rights reserved. Keywords: MrNV and XSV; Susceptibility; Shrimp; RT-PCR; Reinfection
1. Introduction Macrobrachium rosenbergii is the most important and economically cultured palaemonid in the world and it is now farmed in large scale in different parts of the world including India. In 2002, the freshwater prawn production showed a significant increase, ⁎ Corresponding author. Tel.: +91 4172 269487; fax: +91 4172 266487. E-mail address: [email protected] (A.S. Sahul Hameed). 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.02.053
reaching an all time high of 20,000 tonnes in India. Infectious diseases caused by viruses and bacteria constitute the main barrier to the development and continuation of crustacean aquaculture, each cultivated species being sensitive to several types of pathogens. A new viral disease similar to white tail disease (WTD), reported by Arcier et al. (1999) has been observed in freshwater prawn hatcheries and nursery ponds in different parts in India, causing high mortalities and huge economic losses (Sahul Hameed et al., 2004a). Before its occurrence in India, this
R. Sudhakaran et al. / Aquaculture 257 (2006) 136–141
disease was reported from the French West Indies (Arcier et al., 1999), Taiwan (Tung et al., 1999) and China (Qian et al., 2003). The causative agent of WTD was originally reported to be a virus, subsequently identified as M. rosenbergii nodavirus (MrNV) (Arcier et al., 1999). MrNV is a small, icosahedral, non-enveloped virus 26–27nm in diameter. The genome is formed by two pieces of ssRNA (RNA1 and RNA2) of 2.9 and 1.26kb, respectively, and there is a single polypeptide of 43 kDa in the capsid. Qian et al. (2003) subsequently reported the occurrence of an additional extra small virus (XSV) in prawns with WTD collected from China. Sahul Hameed et al. (2004a) have reported the presence of XSV in addition to MrNV in WTD-infected postlarvae of freshwater prawns in India. Various diagnostic methods have been developed to detect these viruses including histopathology, immunological methods, reverse transcriptase-polymerase chain reaction technique (RT-PCR) and in-situ dot blot hybridization method using nucleic acid probes. Romestand and Bonami (2003) have developed a sandwich enzyme-linked immunosorbent assay to detect MrNV in freshwater prawns. Recently, genome-based methods, dot-blot hybridization and RT-PCR have been developed to detect MrNV (Sri Widada et al., 2003) and XSV (Sri Widada et al., 2004; Sahul Hameed et al., 2004a,b). The pathogenicity of these two viral particles in post-larvae and adult freshwater prawns, and distribution of these two viruses in different tissues and organs of experimentally infected prawns have been studied out using RT-PCR assay (Sahul Hameed et al., 2004b). Our previous studies revealed that these viruses failed to infect the adult freshwater prawn and may not be suitable for propagating these viruses in adult prawn (Sahul Hameed et al., 2004b). Lack of established prawn cell lines is also a hurdle in propagating these viruses in large quantity of viruses for various purposes including preparation of diagnostic reagents. We are screening different arthropod species to find out the host range and suitable proliferating system in crustacean species for these viruses. While screening, three species of marine shrimp (Penaeus indicus, Penaeus japonicus and Penaeus monodon) were found to be very useful to proliferate these viruses. Hence, the present study was carried out to examine the infectivity and pathogenicity of the MrNV and XSV to three species of marine shrimp and examine the target organs of these viruses in these shrimp to determine whether these target organs are same as in freshwater prawn. We also studied the possibility of using these marine shrimp to proliferate MrNV and XSV instead of adult freshwater prawn.
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2. Materials and methods 2.1. Preparation of viral inoculum Naturally WTD-infected post-larvae (PL) with prominent sign of whitish muscle in the abdominal region were collected from hatcheries located near Nellore, Andhra Pradesh and used as the source of viral inoculum for infectivity experiments. Frozen infected PL were thawed and homogenized in a sterile homogenizer. A 10% (w/v) suspension was made with TN buffer (20 mM Tris–HCl and 0.4 M NaCl, pH 7.4). The homogenate was centrifuged at 4000×g for 20 min at 4 °C and its supernatant was recentrifuged at 10 000×g for 20min at 4°C before the final supernatant was filtered through a 0.22-μm pore membrane. Then, the presence of MrNV and XSV in tissue suspension was checked by RT-PCR. The filtrate was then stored at − 20 °C for infectivity studies. 2.2. Collection and maintenance of experimental animals P. indicus, P. japonicus and P. monodon (10–15 g body weight) were collected from grow-out ponds or the sea, and maintained in a 1000-l fiberglass tank with an airlift biological filter at room temperature (27–30°C), with salinity between 20 and 25ppt. Natural seawater was used in the experiments. It was pumped from the adjacent sea and allowed to sediment to remove the sand and other suspended particles. The animals were fed with commercial pellet feed (CP shrimp feed, Thailand). Dissolved oxygen, salinity, pH and temperature were measured in alternate days during the experimental period. Salinity was measured with a salinometer and dissolved oxygen was estimated by the Winkler method. From these animals, five per species were randomly selected and screened [gill tissue, stomach, intestine, abdominal tissue (25 mg) or hemolymph (50 μl)] for MrNV and XSV by RT-PCR (Sahul Hameed et al., 2004a). For experimental transmission, healthy post-larvae (PL 10) of M. rosenbergii were collected from a hatchery in a locality with no record of WTD. They were randomly sampled and screened for WTD by RTPCR assay prior to challenge experiments. After collection, the post-larvae were washed with sterile freshwater to remove food and other materials adhering to the body. The washed post-larvae were maintained in glass aquaria (25 l) containing aerated freshwater at a temperature of 27–30 °C and fed twice a day with Artemia nauplii.
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2.3. Infectivity experiments The experimental animals (P. indicus, P. japonicus and P. monodon) (10 per group and tank) were infected by intramuscular injection of MrNV and XSV. Marine shrimp were maintained in 100-l fiberglass tanks at room temperature (27–30 °C) with salinity ranging between 20 and 25ppt. The experimental animals were injected intramuscularly in the second abdominal segment in shrimp with filtrate (300 μg of total protein per animal) prepared from WTD-infected post-larvae of M. rosenbergii using 1 ml insulin syringes. Control animals were injected with tissue filtrate from uninfected post-larvae of freshwater prawn. Tissue samples (gill tissue, stomach, intestine, abdominal tissue and hemolymph) were collected at 4 and 6 days post infection. The samples were stored at − 80 °C for further studies. Each experiment was carried out in triplicates. MrNV and XSV inoculum was prepared from experimental muscle tissue of the shrimp as described above to infect the post-larvae of M. rosenbergii after RT-PCR confirmation to determine the virulence of MrNV and XSV propagated from marine shrimp. For oral route, ten shrimp (P. indicus, P. japonicus or P. monodon) were maintained in aquarium tank 200l and starved for 24 h. The shrimp were fed with MrNV and XSV-infected prawn meat two times per day for 3 days. After the last feeding, the animals were fed with commercial pellet feed. In the control group, the prawn were fed with uninfected prawn meat followed by commercial pellet feed. Tissue samples (gill tissue, stomach, intestine, abdominal tissue and hemolymph) were collected at 3 days post infection. The samples were stored at − 80 °C for RT-PCR analysis. Each experiment was carried out in triplicates. The experimental pathogenicity of inoculum of MrNV and XSV prepared from experimentally MrNV and XSV infected marine shrimp to healthy post-larvae of M. rosenbergii was carried out by immersion challenge. A 10% (w/v) suspension was prepared as mentioned above from gill tissue and muscle of three species of shrimp separately. In the immersion challenge, the post-larvae (30) were placed in beakers (5 L) containing freshwater with continuous aeration. The beakers were covered to prevent contamination. The post-larvae were fed with Artemia nauplii. The viral inoculum (MrNV and XSV) was added to water at a volume equal to 0.1% of the total rearing medium (1mL L− 1) (Venegas et al., 1999; Chen et al., 2000). Control groups were exposed to tissue filtrates (0.1%) prepared from healthy marine shrimp. The experiment was conducted in triplicate.
In all the experiments, the animals were examined twice per day; the number of deaths was recorded and the cumulative mortality levels were calculated. Specimens were collected and RT-PCR was carried out to confirm the presence of MrNV and XSV in the samples. 2.4. Total RNA extraction For extraction of total RNA, tissue samples [gill tissue, stomach, intestine, abdominal tissue (25 mg) or hemolymph (50 μl)] from marine shrimp were collected and homogenized in 300μl of TN buffer (20 mM Tris– HCl, 0.4 M NaCl, pH 7.4). In the case of post-larvae of freshwater prawn, 50mg of whole post-larvae (3–5) was homogenized in TN buffer. The homogenate was centrifuged at 12,000×g for 15min at room temperature (27–30 °C). The supernatant crude tissue extract was extracted using TRIzol reagent (GIBCO-BRL) according to the protocol of the manufacturer. Briefly, 1ml of TRIzol reagent was thoroughly mixed with 150μl of crude tissue extract and incubated at room temperature for 5min before addition of 0.2 ml of chloroform. The sample was vigorously shaken for 2–3 min at room temperature then centrifuged at 12,000×g for 15 min at room temperature. RNA was precipitated from the aqueous phase with isopropanol, washed with 75% ethanol and dissolved in 50μl of TE buffer (10 mM Tris–HCl, 1mM EDTA, pH 7.5). The amount of nucleic acid in the sample was quantified by measuring the absorbance at 260 nm. The purity was checked by measuring the ratio of OD260 nm/OD280 nm. 2.5. RT-PCR and nested RT-PCR for MrNV and XSV RT-PCR was carried out using the Reverse-IT™ 1step RT-PCR kit (ABgene), allowing reverse transcription (RT) and amplification to be performed in a single reaction tube. One pair of primer specific to MrNVRNA2 was designed from sequence data of the MrNV genome (GenBank Accession No. AY222840) and the sequence of the primer is 5′ GCG TTA TAG ATG GCA CAA GG 3′ (Forward) and 5′ AGC TGT GAA ACT TCC ACT GG 3′ (Reverse) (Sahul Hameed et al., 2004a). The size of DNA amplified product is 425 bp. Reactions were performed in 50 μl RT-PCR buffer containing 20 pmol of each primer and RNA template, using the following steps: RT at 52 °C for 30 min; denaturation at 95 °C for 2 min followed by 30 cycles of denaturation at 94 °C for 40s, annealing at 55°C for 40s and elongation at 68 °C for 1min, ending with an additional elongation step of 10min at 68 °C. For XSV detection, the primers were designed in our laboratory
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Fig. 1. Agarose gels showing RT-PCR detection of MrNV and XSV in different organs of experimentally infected marine shrimp, Penaeus monodon. M—DNA marker; 1—virus suspension prepared from infected post-larvae of freshwater; 2—normal post-larvae; 3—gill tissue; 4—hemolymph; 5—abdominal muscle; 6—stomach; 7— intestine.
based on sequence data obtained from GenBank (AY247793) and the sequence is as follows: 5′GGA GAA CCA TGA GAT CAC G 3′ (Forward) and 5′ CTG CTC ATT ACT GTT CGG AGT C 3′ (Reverse) (Sri Widada et al., 2004). The amplification product is 546 bp. The reaction conditions are similar to that for MrNV. The RT-PCR products (10 μl) were then analyzed by electrophoresis on a 1% agarose gel stained with ethidium bromide and visualized by ultraviolet transillumination. 3. Results and discussion The results of experimental transmission carried out on three species of marine shrimp using MrNV and XSV revealed that they were not susceptible to these viruses. MrNV and XSV failed to cause mortality in all the three species of marine shrimp after intramuscular injection of viral inoculum or by oral route by feeding WTD-
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infected meat to shrimp whereas the same viral inoculum caused 100% mortality in post-larvae of M. rosenbergii by immersion challenge after 5 days post infection. RT-PCR analysis on shrimp injected with MrNV and XSV revealed distinct bands of amplified DNA of 425 bp for MrNV and 546 bp for XSV after electrophoresis of the RT-PCR products, and no band was observed in the control group Fig. 1. The same bands were also found in the shrimp fed with WTD-infected prawn meat, but not in control groups fed with uninfected meat. The bands were observed for extracts from gill tissue, hemolymph, abdominal muscle, stomach and intestine (Fig. 1). To confirm the viability and virulence nature of these viruses propagated in marine shrimp, the viral inoculum prepared from tissue homogenates of experimentally MrNV and XSV-injected shrimp was reinoculated by immersion challenge with post-larvae of M. rosenbergii. The inoculum of MrNV and XSV prepared marine shrimp caused 100% mortality within 7 days post infection (Table 1) and moribund post-larvae showed positive for MrNV and XSV by RT-PCR (Fig. 2). The RT-PCR analysis and the data of reinoculation studies confirmed the possibility of marine shrimp acting as potential reservoir for MrNV and XSV in the farming systems. These results also indicate that these viruses are capable of utilizing tissue of marine shrimp as a proliferating system for their propagation and also maintain their virulence. In the present study, the presence of MrNV and XSV in different organs of marine shrimp was confirmed by RT-PCR as observed by Sahul Hameed et al. (2004b) in adult M. rosenbergii. These studies indicate the uniform pattern in the tissue tropism of MrNV and XSV in freshwater prawn and marine shrimp. Knowing pathogen distribution in
Table 1 Cumulative percent mortality of post-larvae of Macrobrachium rosenbergii exposed by immersion challenge to inoculum of MrNV and XSV prepared from different species of marine shrimp Group
No. of individuals used
I II III IV
90 90 90 90
V
90
Viral inoculum from different hosts
Penaneus monodon Penaeus indicus Penaeus japonicus Naturally infected post-larvae of M. rosenbergii Tissue filtrate from normal post-larvae
Cumulative mortality (%) a
WTD detection by RT-PCR
1 day
3 days
5 days
6 days
7 days
0 0 0 0
23.33 0 0 76.67
76.67 43.33 36.67 100
100 73.33 56.67
100 100
0
0
0
0
‘−’ negative RT-PCR reaction; ‘+’ MrNV or XSV positive by RT-PCR. a Experiments were carried out in triplicate.
0
9 days
0
10 days
0
MrNV
XSV
+ + + +
+ + + +
−
−
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Fig. 2. RT-PCR detection of MrNV and XSV in post-larvae of M. rosenbergii exposed to inoculum prepared from different species of marine shrimp. M—DNA marker; Lanes 1 and 6—virus suspension prepared from infected post-larvae of freshwater prawn; Lanes 2 and 7 —healthy post-larvae; Lanes 3 and 8—viral inoculum prepared from P. indicus; Lanes 4 and 9—viral inoculum prepared from P. japonicus; Lanes 5 and 10—viral inoculum prepared from P. monodon.
their sustenance and economic viability. This situation invites the possibility of transmitting pathologically significant organisms from native to non-native hosts as observed in the present study. Based on the results obtained in this study, it is opined that mixed culture of M. rosenbergii with P. monodon is the first thing to be avoided before adopting any preventive measures in the management of WTD. In future, other brackishwater species should be examined thoroughly for the possibility of acting as reservoir or carrier for MrNV and XSV if the opportunity is given by mixed culture with M. rosenbergii either intentionally or by close proximity of the culture environment to each other. Acknowledgements
tissues and organs can help us to understand issues related to disease susceptibility and transmission and to choose optimal samples for pathogen isolation and detection, especially for potential carriers that may require monitoring for control measures. Tissue tropism of WSSV and yellow head virus has been studied by various workers (Lu et al., 1995; Lo et al., 1997; Sahul Hameed et al., 1998) and has proven useful in discovering and closing transmission routes (Lo et al., 1997). Finding both MrNV and XSV in all positive tissues and organs of both freshwater prawn and marine shrimp indicated that the 2 viruses were closely associated, and suggested that they might be mutually dependent as proposed by Qian et al. (2003). MrNV and XSV failed to cause mortality in marine shrimp as observed in adult freshwater prawn by Sahul Hameed et al. (2004b) even though most of the organs in freshwater prawn and marine shrimp showed positive for both viruses. The mechanism of resistance in these animals to MrNV and XSV is not known. Disease resistance in some invertebrates is related to the production of bactericidins, lysins and agglutinins following exposure to foreign proteins (Bang, 1967; Mckay and Jenkin, 1969). It is possible that similar substances may account for the resistance of these animals to MrNV and XSV. Prevailing low market price for shrimp and occurrence of white spot syndrome made the farmers to think for a viable alternate culture in low saline coastal areas. The only option available to the farmers is freshwater prawn M. rosenbergii. Unfortunately an area specific (Nellore, Andhra Pradesh) problem in the form of appendage deformities is affecting freshwater prawn production and its survival (Ravi Kumar et al., 2004). Under these circumstances some percentage of farmers have considered either mixed culture of shrimp (P. monodon) with M. rosenbergii or by crop rotation between these two species as a viable alternative for
The authors are thankful to the Management of C. Abdul Hakeem College, Melvisharam for providing the facilities to carry out this work. This study was funded by a grant from Indian Council of Agricultural Research, Government of India and IFCPAR, New Delhi. References Arcier, J.M., Herman, F., Lightner, D.V., Redman, R., Mari, J., Bonami, J.R., 1999. A viral disease associated with mortalities in hatchery-reared postlarvae of the giant freshwater prawn Macrobrachium rosenbergii. Dis. Aquat. Org. 38, 177–181. Bang, F.B., 1967. Serological responses among invertebrates other than insects. Fed. Proc. 267, 1680–1684. Chen, L.L., Lo, C.F., Chiu, Y.L., Chang, C.F., Kou, G.H., 2000. Natural and experimental infection of white spot syndrome virus WSSV in benthic larvae of mud crab Scylla serrata. Dis. Aquat. Org. 40, 157–161. Lo, C.F., Ho, C.H., Chen, C.H., Liu, K.F., Liu, Y.L., Yeh, P.Y., Peng, S. E., Hsu, H.C., Liu, H.C., Chang, C.F., Su, M.S., Wang, C.H., Kou, G.H., 1997. Detection and tissue tropism of white spot syndrome baculovirus (WSSV) in captured brooders of Penaeus monodon with special emphasis on reproductive organs. Dis. Aquat. Org. 30, 53–72. Lu, Y., Tapay, L.M., Loh, P.C., Brock, J.A., 1995. Infection of the yellow head baculo-like virus in two species of Penaeid shrimp, P. stylirostris and P. vannamei. J. Fish Dis. 17, 649–656. Mckay, D., Jenkin, C.R., 1969. Immunity in invertebrates: II. Adaptive immunity in the cray fish Parachaeraps bicarinatu. Immunology 17, 127–137. Qian, D., Shi, Z., Zhang, S., Cao, Z., Liu, W., Li, L., Xie, Y., Cambournac, I., Bonami, J.R., 2003. Extra small virus-like particles (XSV) and nodavirus associated with whitish muscle disease in the giant freshwater prawn, Macrobrachium rosenbergii. J. Fish Dis. 26, 521–527. Ravi Kumar, A., Venkateswara Rao, G., Sambasiva Rao, K.R.S., 2004. Dis. Aquat. Org. 59, 75–78. Romestand, B., Bonami, J.R., 2003. A sandwich enzyme linked immunosorbent assay (S-ELISA) for detection of MrNV in the giant freshwater prawn, Macrobrachium rosenbergii (de Man). J. Fish Dis. 26, 71–75.
R. Sudhakaran et al. / Aquaculture 257 (2006) 136–141 Sahul Hameed, A.S., Anilkumar, M., Stephen Raj, M.L., Kunthala Jayaraman, 1998. Studies on the pathogencity of systemic ectodermal and mesodermal baculovirus and its detection in shrimp by immunological methods. Aquaculture 160, 31–45. Sahul Hameed, A.S., Yoganandhan, K., Widada, J., Bonami, J.R., 2004a. Studies on the occurrence and RT-PCR detection of Macrobrachium rosenbergii nodavirus and extra small virus-like particles associated with white tail disease of Macrobrachium rosenbergii in India. Aquaculture 238, 127–133. Sahul Hameed, A.S., Yoganandhan, K., Widada, J., Bonami, J.R., 2004b. Experimental transmission and tissue tropism of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus like-particles in Macrobrachium rosenbergii. Dis. Aquat. Org. 62, 191–196. Widada, J., Durand, S., Cambournac, Qian, D., Shi, Z., Dejonghe, E., Richard, V., Bonami, J.R., 2003. Genome-based detection methods of Macrobrachium rosenbergii nodavirus, a pathogen of the giant
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freshwater prawn, Macrobrachium rosenbergii: dot-blot, in situ hybridization and RT-PCR. J. Fish Dis. 26, 583–590. Widada, J., Richard, V., Cambournac, Shi, Z., Qian, D., Bonami, J.R., 2004. Dot-blot hybridization and RT-PCR detection of extra small virus (XSV) associated with white tail disease of prawn Macrobrachium rossenbergii. Dis. Aquat. Org. 58, 83–87. Tung, C.W., Wang, C.S., Chen, S.N., 1999. Histological and electron microscopic study on Macrobrachium muscle virus (MMV) infection in the giant freshwater prawn, Macrobrachium rosenbergii (de Man), cultured in Taiwan. J. Fish Dis. 22, 1–5. Venegas, C.A., Nonaka, L., Mushiake, K., Nishizawa, T., Muroga, K., 1999. Pathogenicity of penaeid rod-shaped DNA virus PRDV to kuruma prawn at different development stages. Fish Pathol. 170, 179–194.
Experimental transmission of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) in three species of marine shrimp (Penaeus indicus, Penaeus japonicus and Penaeus monodon) R. Sudhakaran a , S. Syed Musthaq a , P. Haribabu b , S.C. Mukherjee b , C. Gopal c , A.S. Sahul Hameed a,⁎ a
Department of Zoology, C.Abdul Hakeem College, Melvisharam-632 509, Vellore Dist., Tamil Nadu, India b Central Institute of Fisheries Education, Fisheries University Road, Versova, Mumbai-400 061, India c Central Institute of Brackishwater Aquaclture, Santhom High Road, R.A. Puram, Chennai-600 028, India Received 27 September 2005; received in revised form 21 February 2006; accepted 22 February 2006
Abstract The susceptibility of three species of marine shrimp (Penaeus indicus, Penaeus japonicus and Penaeus monodon) to Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) was tested by oral route and intramuscular injection. The results revealed that these marine shrimp were not susceptible to these viruses which failed to produce mortality in shrimp. RTPCR analysis revealed the presence of MrNV and XSV in different organs such as gill, abdominal muscle, stomach, intestine and hemolymph of three species of shrimp injected with viruses. These viruses were also found in different tissues of shrimp fed with WTD-infected prawn meat, but not in control groups fed with uninfected meat. The reinoculation studies using the inoculum of MrNV and XSV prepared from marine shrimp caused 100% mortality in the post-larvae of freshwater prawn and the moribund post-larvae showed positive for these viruses by RT-PCR. The results of present study indicate the possibility of marine shrimp acting as reservoir for MrNV and XSV and maintaining their virulence in tissue system of marine shrimp. © 2006 Elsevier B.V. All rights reserved. Keywords: MrNV and XSV; Susceptibility; Shrimp; RT-PCR; Reinfection
1. Introduction Macrobrachium rosenbergii is the most important and economically cultured palaemonid in the world and it is now farmed in large scale in different parts of the world including India. In 2002, the freshwater prawn production showed a significant increase, ⁎ Corresponding author. Tel.: +91 4172 269487; fax: +91 4172 266487. E-mail address: [email protected] (A.S. Sahul Hameed). 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.02.053
reaching an all time high of 20,000 tonnes in India. Infectious diseases caused by viruses and bacteria constitute the main barrier to the development and continuation of crustacean aquaculture, each cultivated species being sensitive to several types of pathogens. A new viral disease similar to white tail disease (WTD), reported by Arcier et al. (1999) has been observed in freshwater prawn hatcheries and nursery ponds in different parts in India, causing high mortalities and huge economic losses (Sahul Hameed et al., 2004a). Before its occurrence in India, this
R. Sudhakaran et al. / Aquaculture 257 (2006) 136–141
disease was reported from the French West Indies (Arcier et al., 1999), Taiwan (Tung et al., 1999) and China (Qian et al., 2003). The causative agent of WTD was originally reported to be a virus, subsequently identified as M. rosenbergii nodavirus (MrNV) (Arcier et al., 1999). MrNV is a small, icosahedral, non-enveloped virus 26–27nm in diameter. The genome is formed by two pieces of ssRNA (RNA1 and RNA2) of 2.9 and 1.26kb, respectively, and there is a single polypeptide of 43 kDa in the capsid. Qian et al. (2003) subsequently reported the occurrence of an additional extra small virus (XSV) in prawns with WTD collected from China. Sahul Hameed et al. (2004a) have reported the presence of XSV in addition to MrNV in WTD-infected postlarvae of freshwater prawns in India. Various diagnostic methods have been developed to detect these viruses including histopathology, immunological methods, reverse transcriptase-polymerase chain reaction technique (RT-PCR) and in-situ dot blot hybridization method using nucleic acid probes. Romestand and Bonami (2003) have developed a sandwich enzyme-linked immunosorbent assay to detect MrNV in freshwater prawns. Recently, genome-based methods, dot-blot hybridization and RT-PCR have been developed to detect MrNV (Sri Widada et al., 2003) and XSV (Sri Widada et al., 2004; Sahul Hameed et al., 2004a,b). The pathogenicity of these two viral particles in post-larvae and adult freshwater prawns, and distribution of these two viruses in different tissues and organs of experimentally infected prawns have been studied out using RT-PCR assay (Sahul Hameed et al., 2004b). Our previous studies revealed that these viruses failed to infect the adult freshwater prawn and may not be suitable for propagating these viruses in adult prawn (Sahul Hameed et al., 2004b). Lack of established prawn cell lines is also a hurdle in propagating these viruses in large quantity of viruses for various purposes including preparation of diagnostic reagents. We are screening different arthropod species to find out the host range and suitable proliferating system in crustacean species for these viruses. While screening, three species of marine shrimp (Penaeus indicus, Penaeus japonicus and Penaeus monodon) were found to be very useful to proliferate these viruses. Hence, the present study was carried out to examine the infectivity and pathogenicity of the MrNV and XSV to three species of marine shrimp and examine the target organs of these viruses in these shrimp to determine whether these target organs are same as in freshwater prawn. We also studied the possibility of using these marine shrimp to proliferate MrNV and XSV instead of adult freshwater prawn.
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2. Materials and methods 2.1. Preparation of viral inoculum Naturally WTD-infected post-larvae (PL) with prominent sign of whitish muscle in the abdominal region were collected from hatcheries located near Nellore, Andhra Pradesh and used as the source of viral inoculum for infectivity experiments. Frozen infected PL were thawed and homogenized in a sterile homogenizer. A 10% (w/v) suspension was made with TN buffer (20 mM Tris–HCl and 0.4 M NaCl, pH 7.4). The homogenate was centrifuged at 4000×g for 20 min at 4 °C and its supernatant was recentrifuged at 10 000×g for 20min at 4°C before the final supernatant was filtered through a 0.22-μm pore membrane. Then, the presence of MrNV and XSV in tissue suspension was checked by RT-PCR. The filtrate was then stored at − 20 °C for infectivity studies. 2.2. Collection and maintenance of experimental animals P. indicus, P. japonicus and P. monodon (10–15 g body weight) were collected from grow-out ponds or the sea, and maintained in a 1000-l fiberglass tank with an airlift biological filter at room temperature (27–30°C), with salinity between 20 and 25ppt. Natural seawater was used in the experiments. It was pumped from the adjacent sea and allowed to sediment to remove the sand and other suspended particles. The animals were fed with commercial pellet feed (CP shrimp feed, Thailand). Dissolved oxygen, salinity, pH and temperature were measured in alternate days during the experimental period. Salinity was measured with a salinometer and dissolved oxygen was estimated by the Winkler method. From these animals, five per species were randomly selected and screened [gill tissue, stomach, intestine, abdominal tissue (25 mg) or hemolymph (50 μl)] for MrNV and XSV by RT-PCR (Sahul Hameed et al., 2004a). For experimental transmission, healthy post-larvae (PL 10) of M. rosenbergii were collected from a hatchery in a locality with no record of WTD. They were randomly sampled and screened for WTD by RTPCR assay prior to challenge experiments. After collection, the post-larvae were washed with sterile freshwater to remove food and other materials adhering to the body. The washed post-larvae were maintained in glass aquaria (25 l) containing aerated freshwater at a temperature of 27–30 °C and fed twice a day with Artemia nauplii.
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2.3. Infectivity experiments The experimental animals (P. indicus, P. japonicus and P. monodon) (10 per group and tank) were infected by intramuscular injection of MrNV and XSV. Marine shrimp were maintained in 100-l fiberglass tanks at room temperature (27–30 °C) with salinity ranging between 20 and 25ppt. The experimental animals were injected intramuscularly in the second abdominal segment in shrimp with filtrate (300 μg of total protein per animal) prepared from WTD-infected post-larvae of M. rosenbergii using 1 ml insulin syringes. Control animals were injected with tissue filtrate from uninfected post-larvae of freshwater prawn. Tissue samples (gill tissue, stomach, intestine, abdominal tissue and hemolymph) were collected at 4 and 6 days post infection. The samples were stored at − 80 °C for further studies. Each experiment was carried out in triplicates. MrNV and XSV inoculum was prepared from experimental muscle tissue of the shrimp as described above to infect the post-larvae of M. rosenbergii after RT-PCR confirmation to determine the virulence of MrNV and XSV propagated from marine shrimp. For oral route, ten shrimp (P. indicus, P. japonicus or P. monodon) were maintained in aquarium tank 200l and starved for 24 h. The shrimp were fed with MrNV and XSV-infected prawn meat two times per day for 3 days. After the last feeding, the animals were fed with commercial pellet feed. In the control group, the prawn were fed with uninfected prawn meat followed by commercial pellet feed. Tissue samples (gill tissue, stomach, intestine, abdominal tissue and hemolymph) were collected at 3 days post infection. The samples were stored at − 80 °C for RT-PCR analysis. Each experiment was carried out in triplicates. The experimental pathogenicity of inoculum of MrNV and XSV prepared from experimentally MrNV and XSV infected marine shrimp to healthy post-larvae of M. rosenbergii was carried out by immersion challenge. A 10% (w/v) suspension was prepared as mentioned above from gill tissue and muscle of three species of shrimp separately. In the immersion challenge, the post-larvae (30) were placed in beakers (5 L) containing freshwater with continuous aeration. The beakers were covered to prevent contamination. The post-larvae were fed with Artemia nauplii. The viral inoculum (MrNV and XSV) was added to water at a volume equal to 0.1% of the total rearing medium (1mL L− 1) (Venegas et al., 1999; Chen et al., 2000). Control groups were exposed to tissue filtrates (0.1%) prepared from healthy marine shrimp. The experiment was conducted in triplicate.
In all the experiments, the animals were examined twice per day; the number of deaths was recorded and the cumulative mortality levels were calculated. Specimens were collected and RT-PCR was carried out to confirm the presence of MrNV and XSV in the samples. 2.4. Total RNA extraction For extraction of total RNA, tissue samples [gill tissue, stomach, intestine, abdominal tissue (25 mg) or hemolymph (50 μl)] from marine shrimp were collected and homogenized in 300μl of TN buffer (20 mM Tris– HCl, 0.4 M NaCl, pH 7.4). In the case of post-larvae of freshwater prawn, 50mg of whole post-larvae (3–5) was homogenized in TN buffer. The homogenate was centrifuged at 12,000×g for 15min at room temperature (27–30 °C). The supernatant crude tissue extract was extracted using TRIzol reagent (GIBCO-BRL) according to the protocol of the manufacturer. Briefly, 1ml of TRIzol reagent was thoroughly mixed with 150μl of crude tissue extract and incubated at room temperature for 5min before addition of 0.2 ml of chloroform. The sample was vigorously shaken for 2–3 min at room temperature then centrifuged at 12,000×g for 15 min at room temperature. RNA was precipitated from the aqueous phase with isopropanol, washed with 75% ethanol and dissolved in 50μl of TE buffer (10 mM Tris–HCl, 1mM EDTA, pH 7.5). The amount of nucleic acid in the sample was quantified by measuring the absorbance at 260 nm. The purity was checked by measuring the ratio of OD260 nm/OD280 nm. 2.5. RT-PCR and nested RT-PCR for MrNV and XSV RT-PCR was carried out using the Reverse-IT™ 1step RT-PCR kit (ABgene), allowing reverse transcription (RT) and amplification to be performed in a single reaction tube. One pair of primer specific to MrNVRNA2 was designed from sequence data of the MrNV genome (GenBank Accession No. AY222840) and the sequence of the primer is 5′ GCG TTA TAG ATG GCA CAA GG 3′ (Forward) and 5′ AGC TGT GAA ACT TCC ACT GG 3′ (Reverse) (Sahul Hameed et al., 2004a). The size of DNA amplified product is 425 bp. Reactions were performed in 50 μl RT-PCR buffer containing 20 pmol of each primer and RNA template, using the following steps: RT at 52 °C for 30 min; denaturation at 95 °C for 2 min followed by 30 cycles of denaturation at 94 °C for 40s, annealing at 55°C for 40s and elongation at 68 °C for 1min, ending with an additional elongation step of 10min at 68 °C. For XSV detection, the primers were designed in our laboratory
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Fig. 1. Agarose gels showing RT-PCR detection of MrNV and XSV in different organs of experimentally infected marine shrimp, Penaeus monodon. M—DNA marker; 1—virus suspension prepared from infected post-larvae of freshwater; 2—normal post-larvae; 3—gill tissue; 4—hemolymph; 5—abdominal muscle; 6—stomach; 7— intestine.
based on sequence data obtained from GenBank (AY247793) and the sequence is as follows: 5′GGA GAA CCA TGA GAT CAC G 3′ (Forward) and 5′ CTG CTC ATT ACT GTT CGG AGT C 3′ (Reverse) (Sri Widada et al., 2004). The amplification product is 546 bp. The reaction conditions are similar to that for MrNV. The RT-PCR products (10 μl) were then analyzed by electrophoresis on a 1% agarose gel stained with ethidium bromide and visualized by ultraviolet transillumination. 3. Results and discussion The results of experimental transmission carried out on three species of marine shrimp using MrNV and XSV revealed that they were not susceptible to these viruses. MrNV and XSV failed to cause mortality in all the three species of marine shrimp after intramuscular injection of viral inoculum or by oral route by feeding WTD-
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infected meat to shrimp whereas the same viral inoculum caused 100% mortality in post-larvae of M. rosenbergii by immersion challenge after 5 days post infection. RT-PCR analysis on shrimp injected with MrNV and XSV revealed distinct bands of amplified DNA of 425 bp for MrNV and 546 bp for XSV after electrophoresis of the RT-PCR products, and no band was observed in the control group Fig. 1. The same bands were also found in the shrimp fed with WTD-infected prawn meat, but not in control groups fed with uninfected meat. The bands were observed for extracts from gill tissue, hemolymph, abdominal muscle, stomach and intestine (Fig. 1). To confirm the viability and virulence nature of these viruses propagated in marine shrimp, the viral inoculum prepared from tissue homogenates of experimentally MrNV and XSV-injected shrimp was reinoculated by immersion challenge with post-larvae of M. rosenbergii. The inoculum of MrNV and XSV prepared marine shrimp caused 100% mortality within 7 days post infection (Table 1) and moribund post-larvae showed positive for MrNV and XSV by RT-PCR (Fig. 2). The RT-PCR analysis and the data of reinoculation studies confirmed the possibility of marine shrimp acting as potential reservoir for MrNV and XSV in the farming systems. These results also indicate that these viruses are capable of utilizing tissue of marine shrimp as a proliferating system for their propagation and also maintain their virulence. In the present study, the presence of MrNV and XSV in different organs of marine shrimp was confirmed by RT-PCR as observed by Sahul Hameed et al. (2004b) in adult M. rosenbergii. These studies indicate the uniform pattern in the tissue tropism of MrNV and XSV in freshwater prawn and marine shrimp. Knowing pathogen distribution in
Table 1 Cumulative percent mortality of post-larvae of Macrobrachium rosenbergii exposed by immersion challenge to inoculum of MrNV and XSV prepared from different species of marine shrimp Group
No. of individuals used
I II III IV
90 90 90 90
V
90
Viral inoculum from different hosts
Penaneus monodon Penaeus indicus Penaeus japonicus Naturally infected post-larvae of M. rosenbergii Tissue filtrate from normal post-larvae
Cumulative mortality (%) a
WTD detection by RT-PCR
1 day
3 days
5 days
6 days
7 days
0 0 0 0
23.33 0 0 76.67
76.67 43.33 36.67 100
100 73.33 56.67
100 100
0
0
0
0
‘−’ negative RT-PCR reaction; ‘+’ MrNV or XSV positive by RT-PCR. a Experiments were carried out in triplicate.
0
9 days
0
10 days
0
MrNV
XSV
+ + + +
+ + + +
−
−
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Fig. 2. RT-PCR detection of MrNV and XSV in post-larvae of M. rosenbergii exposed to inoculum prepared from different species of marine shrimp. M—DNA marker; Lanes 1 and 6—virus suspension prepared from infected post-larvae of freshwater prawn; Lanes 2 and 7 —healthy post-larvae; Lanes 3 and 8—viral inoculum prepared from P. indicus; Lanes 4 and 9—viral inoculum prepared from P. japonicus; Lanes 5 and 10—viral inoculum prepared from P. monodon.
their sustenance and economic viability. This situation invites the possibility of transmitting pathologically significant organisms from native to non-native hosts as observed in the present study. Based on the results obtained in this study, it is opined that mixed culture of M. rosenbergii with P. monodon is the first thing to be avoided before adopting any preventive measures in the management of WTD. In future, other brackishwater species should be examined thoroughly for the possibility of acting as reservoir or carrier for MrNV and XSV if the opportunity is given by mixed culture with M. rosenbergii either intentionally or by close proximity of the culture environment to each other. Acknowledgements
tissues and organs can help us to understand issues related to disease susceptibility and transmission and to choose optimal samples for pathogen isolation and detection, especially for potential carriers that may require monitoring for control measures. Tissue tropism of WSSV and yellow head virus has been studied by various workers (Lu et al., 1995; Lo et al., 1997; Sahul Hameed et al., 1998) and has proven useful in discovering and closing transmission routes (Lo et al., 1997). Finding both MrNV and XSV in all positive tissues and organs of both freshwater prawn and marine shrimp indicated that the 2 viruses were closely associated, and suggested that they might be mutually dependent as proposed by Qian et al. (2003). MrNV and XSV failed to cause mortality in marine shrimp as observed in adult freshwater prawn by Sahul Hameed et al. (2004b) even though most of the organs in freshwater prawn and marine shrimp showed positive for both viruses. The mechanism of resistance in these animals to MrNV and XSV is not known. Disease resistance in some invertebrates is related to the production of bactericidins, lysins and agglutinins following exposure to foreign proteins (Bang, 1967; Mckay and Jenkin, 1969). It is possible that similar substances may account for the resistance of these animals to MrNV and XSV. Prevailing low market price for shrimp and occurrence of white spot syndrome made the farmers to think for a viable alternate culture in low saline coastal areas. The only option available to the farmers is freshwater prawn M. rosenbergii. Unfortunately an area specific (Nellore, Andhra Pradesh) problem in the form of appendage deformities is affecting freshwater prawn production and its survival (Ravi Kumar et al., 2004). Under these circumstances some percentage of farmers have considered either mixed culture of shrimp (P. monodon) with M. rosenbergii or by crop rotation between these two species as a viable alternative for
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