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Penaeus monodon nucleopolyhedrovirus detection using an immunochromatographic strip test
Journal of Virological Methods 183 (2012) 210–214
Contents lists available at SciVerse ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Penaeus monodon nucleopolyhedrovirus detection using an immunochromatographic strip test Pradit Wangman a , Siwaporn Longyant a , Parin Chaivisuthangkura a , Pattarin Sridulyakul a , Sombat Rukpratanporn b , Paisarn Sithigorngul a,∗ a b
Department of Biology, Srinakharinwirot University, Bangkok 10110, Thailand Center of Excellence for Marine Biotechnology at Chulalongkorn University, National Center for Genetic Engineering and Biotechnology (BIOTEC), Bangkok 10330, Thailand
a b s t r a c t Article history: Received 8 December 2011 Received in revised form 3 April 2012 Accepted 30 April 2012 Available online 10 May 2012 Keywords: Immunochromatographic strip test Monoclonal antibody Polyhedrin Penaeus monodon nucleopolyhedrovirus (PemoNPV) Monodon baculovirus (MBV)
An immunochromatographic strip test is described for detection of the polyhedrin protein of Penaeus monodon nucleopolyhedrovirus (PemoNPV). The test employs one monoclonal antibody (MAb MBV5) conjugated to colloidal gold to bind to polyhedrin protein and a 1:1:1 mixture of 3 other MAbs (MBV8, 14 and 21) to capture colloidal-gold MAb–protein complexes at a test (T) line on the nitrocellulose strip. A downstream control (C) line of goat anti-mouse immunoglobulin G (GAM) antibody is used to capture excess free colloidal-gold conjugated MBV5 to validate test performance. Heating of homogenates of PemoNPV-infected P. monodon postlarvae prepared in PBS for 30 min was necessary to maximize T line color intensity, and homogenates of infected postlarvae could still be scored as PemoNPV-positive when diluted 1:64. A strip test result was obtained within 15 min of sample application, and although about 200-fold lower than a one-step PCR test for PemoNPV, its detection sensitivity was comparable to a dot blot. Due to its simplicity not reliant on sophisticated equipment or specialized skills, the strip test could be adopted to screen easily for PemoNPV infections at shrimp hatcheries and farms. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Penaeus monodon nucleopolyhedrovirus (PemoNPV) or monodon baculovirus (MBV) as it has been called previously, occurs commonly as a subclinical infection in black tiger (P. monodon) shrimp broodstock but can cause disease in early life stages (larvae, protozoea, mysis, postlarvae) or compromise their resilience against Vibrio spp. and other protozoa (Ramasamy et al., 2000; Vaseeharan and Ramasamy, 2003). Although PemoNPV infection is not associated generally with mortalities in farmed P. monodon (Fegan et al., 1991), it has been linked circumstantially with stunting (Flegel et al., 1999, 2004; Rai et al., 2009). PemoNPV infection can be diagnosed using squash mounts of shrimp hepatopancreas tissue and malachite green to stain intranuclear occlusion bodies or using histology to identify cells with enlarged nuclei containing acidophilic occlusions (Flegel, 2006). Polymerase chain reaction (PCR) tests (Chang et al., 1993; Lu et al., 1993; Belcher and Young, 1998; Hsu et al., 2000) and in situ hybridization assays on histological sections are also available to detect PemoNPV (Poulos et al., 1994). Although the PCR method described by Belcher and Young (1998) proved more reliable for detecting PemoNPV strains in shrimp from Australia, India and
Thailand (Flegel, 2006), a revised PCR test has since been proven to be useful for detecting strains from ten geographically disparate regions (Surachetpong et al., 2005). Real-time PCR (Yan et al., 2009) and loop-mediated isothermal amplification (LAMP) methods (Chaivisuthangkura et al., 2009; Nimitphak et al., 2010) have also been shown to be effective in detecting PemoNPV strains from Thailand and Indonesia. Methods using polyclonal or monoclonal antibodies (MAbs) have been developed for the detection of several shrimp pathogens including PemoNPV (Satidkanitkul et al., 2005; Boonsanongchokying et al., 2006; Sridulyakul et al., 2011). Although less sensitive than PCR, such antibody-based diagnostic methods tend to be simpler, less expensive and more amenable to use in less well equipped laboratories or by unskilled personnel in the field (Powell et al., 2006; Sithigorngul et al., 2006, 2007, 2011). Here, a simple immunochromatographic strip test method able to detect rapidly PemoNPV in P. monodon postlarvae is described that employs a set of four MAbs specific to the PemoNPV polyhedrin protein (Boonsanongchokying et al., 2006).
2. Materials and methods 2.1. Virus
∗ Corresponding author. Tel.: +66 2 664 1000x8515; fax: +66 2 260 0127. E-mail addresses: paisarn [email protected], [email protected] (P. Sithigorngul). 0166-0934/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2012.04.016
PemoNPV-infection in P. monodon postlarvae 10 and postlarvae 15 collected from a farm in Chonburi province, Thailand
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was verified using one-step PCR (Surachetpong et al., 2005). Individual postlarvae were homogenized in 50 l PBS (0.15 M phosphate buffered saline, pH 7.2), heated at 100 ◦ C for 30 min and homogenate aliquots were stored at −20 ◦ C. 2.2. Monoclonal antibody preparation The four monoclonal antibodies (MAbs MBV5, 8, 14, and 21) used in the strip test are reactive to different epitopes on the PemoNPV polyhedrin protein and were produced as described previously from a mouse immunized with the native protein (Boonsanongchokying et al., 2006). MAbs secreted from hybridoma cells grown in serum-free Hybridoma-SFM media (Gibco, Grand Island, NY, USA) were purified using Protein G-agarose columns (Roche Molecular Biochemicals, Indianapolis, IN, USA) according to the manufacturer’s instructions. The MAbs were dialyzed against 10 mM phosphate buffer, pH 7.3 and adjusted to a concentration of 1 mg/ml before being stored in aliquots at −20 ◦ C. 2.3. Dot blot Aliquots of 2-fold dilution series of homogenates of uninfected postlarvae 10 or PemoNPV-infected postlarvae 15 were spotted (1 l/spot) onto nitrocellulose membranes and baked at 60 ◦ C for 10 min. After incubating for 4 h in various hybridoma culture media diluted 1:20 in 5% blocking solution (5% nonfat dry milk and 0.1% Triton X-100 in PBS), bound MAbs were detected by incubation for 3 h in 1:1500 horseradish peroxidase-labeled goat anti-mouse gamma immunoglobulin heavy and light chain-specific antibody (GAM-HRP, Bio-Rad, Hercules, CA, USA) followed by incubation in 0.03% diaminobenzidine (DAB) substrate in 0.006% H2 O2 , 0.05% cobalt chloride in PBS as described previously (Sithigorngul et al., 2000). 2.4. Immunochromatographic test strip The layout, assembly and operation of the immunochromatographic test strip based on 8 m pore-size nitrocellulose (NC) membranes (Pacific Biotech Co. Ltd., Thailand) have been described in detail previously (Sithigorngul et al., 2011). Briefly, the MAb MBV5 used to capture PemoNPV polyhedrin protein from samples was conjugated to 10 nm diameter colloidal gold particles and sprayed (3 l/cm) onto a glass fiber pad and dried overnight at 40 ◦ C. On the NC membrane, a 1:1:1 mixture of 1 mg/ml MAbs MBV8, MBV14 and MBV21 was micro-sprayed (1 l/cm) at the test (T) line position and goat anti-mouse IgG antibody (0.8 mg/ml) was micro-sprayed (1 l/cm) at the control (C) line position, and the membranes were then dried overnight at 40 ◦ C. The pad containing gold-conjugated MBV5 was placed between the well for sample application and the T line, and an absorption pad was placed downstream of the C line. The assembled test strips were stored desiccated in a plastic bag until used. 2.5. Optimizing sample preparation for strip test analysis As PemoNPV polyhedrin protein aggregate to form dense occlusion bodies in the nucleus, antibody access is limited to the occlusion body surface. To optimize occlusion body disruption, homogenates of individual postlarvae 15 diluted in 50 l PBS or application buffer (30 mM Tris, 336 mM NaCl, 9 nM EDTA, 1% Triton X-100, pH 9.3) were heated at 100 ◦ C for various time periods (0, 5, 15, 30 and 60 min). After mixing with an equal volume of application buffer, 100 l homogenate was applied to the strip test. To identify whether negative detections with postlarvae homogenized and heated in application buffer were not due to the polyhedrin protein degradation, homogenates diluted in PBS
Fig. 1. PemoNPV strip test results using homogenates of (A) PemoNPV-infected P. monodon postlarvae 15 and (B) uninfected postlarvae 10. (For interpretation of the references to color in the text, the reader is referred to the web version of the article.)
or application buffer and heated for 60 min were subjected to SDS-PAGE and Western blot analysis as described previously (Sridulyakul et al., 2011) using MAb MBV8. 2.6. Specificity testing Individual uninfected or PemoNPV-infected P. monodon postlarvae 8, postlarvae 10 and postlarvae 15 were homogenized in 50 l PBS, boiled for 30 min, mixed 1:1 with application buffer and analyzed in the strip test. Reddish-purple colored bands developed within 15 min at both T and C lines when PemoNPV was detected but only at the C line when it was not detected (Fig. 1). To assess strip test specificity against common shrimp viruses, homogenates of pleopods from shrimp infected with white spot syndrome virus (WSSV), yellow head virus (YHV), Taura syndrome virus (TSV) or Penaeus stylirostris densovirus (PstDNV) were applied. 2.7. Strip test sensitivity compared to dot blot and PCR Individual uninfected P. monodon postlarvae 10 or PemoNPVinfected postlarvae 15 were homogenized in 50 l PBS and heated at 100 ◦ C for 30 min. Aliquots (1 l) of this homogenate were used directly to prepare dilution series for dot blot analysis or mixed 1:1 in application buffer before analysis using the strip test or and PCR. Nucleic acid extracted from 100 l homogenate using a High Pure Viral Nucleic Acid Kit (Roche Molecular Biochemicals) and diluted serially in 50 ng/l nucleic acid extracted from uninfected shrimp was tested using a PemoNPV PCR method described previously (Surachetpong et al., 2005). Aliquots (100 l) of these serial dilutions were also applied to immunochromatographic test strips. 2.8. Thermostability of the strip test To assess thermostability, immunochromatographic test strips in a desiccated plastic bag were placed in a 60 ◦ C oven for 10, 20 or 30 days before use with homogenates of uninfected or PemoNPVinfected postlarvae diluted 1:5 or 1:50 in application buffer (10 strips tested per time point). 3. Results and discussion 3.1. Strip test optimization In the optimized strip test method, four MAbs targeted to different epitopes were combined with different combinations and used for colloidal gold conjugate or test line. Finally MAbs were selected for gold-labeling and initial binding (MBV5) and
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Fig. 2. Optimization of sample preparation methods for the PemoNPV strip test. Postlarvae homogenized in (A) PBS or (B) application buffer were heated at 100 ◦ C for 0, 5, 15, 30 and 60 min. Postlarvae homogenized in PBS and application buffer and heated for 60 min (arrowheads) were also subjected to SDS-PAGE and Western blot analysis to detect PemoNPV polyhedrin protein (58 kDa). (For interpretation of the references to color in the text, the reader is referred to the web version of the article.)
capture of PemoNPV polyhedrin protein (MAbs MBV8, MBV14, and MBV21) based of this combination resulting in the highest band color intensity and sensitivity based on detection of serial dilutions of PemoNPV-infected P. monodon postlarvae homogenates without giving false-positives with homogenates prepared from uninfected postlarvae (Fig. 1).
3.2. Sample preparation method Among individual PemoNPV-infected postlarvae homogenized in PBS, no band resulted without heating, heating at 100 ◦ C for 5 min resulted in a lightly reddish purple colored band and heating for 15, 30 or 60 min resulted in darkly reddish purple colored bands
Fig. 3. Sensitivity limit of the strip test and dot blots for detecting PemoNPV. (A) Strip test detection of serial 2-fold dilutions of PemoNPV-infected postlarvae 15 homogenized in PBS, (B) dot blot detection of serial 2-fold dilutions of postlarvae homogenate using each single MAb or all 4 MAbs combined. N = uninfected postlarvae homogenate. Arrowheads indicate the lowest amount detected reliably.
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3.5. Strip test thermostability Using strip tests stored desiccated in a 60 ◦ C oven for various time periods, PemoNPV-infected postlarvae homogenates diluted 1:5 were detected equally well with strips stored for up to 20 days, but slightly less well using strips stored for 30 days (data not shown). With PemoNPV-infected postlarvae homogenates diluted 1:50, the color intensity of positive bands was comparable with strips stored at 60 ◦ C for up to 30 days. Data obtained with strips heated at this temperature indicate that the strip tests should remain functional for up to 2 years when stored at room temperature (Paek et al., 2000), as found similarly for the strip tests for WSSV and YHV (Sithigorngul et al., 2006, 2007, 2011). 4. Conclusions Fig. 4. One-step PCR detection of PemoNPV DNA in nucleic acid extracted from PemoNPV-infected postlarvae 15 from Fig. 3 and serially diluted 10-fold in nucleic acid from an uninfected postlarvae. Lane M: DNA markers; Lanes (a) 10−1 , (b) 10−2 , (c) 10−3 , (d) 10−4 , (e) 10−5 dilutions, and (f) uninfected postlarvae homogenate. The arrowhead indicates the detection limit of the 261 bp PCR product.
similar in intensity (Fig. 2A). Among postlarvae homogenized in application buffer, no band also resulted without heating, and heating for any length of time only resulted in a lightly gray colored band (Fig. 2B) of non-specific reaction. To confirm that the polyhedrin protein had not degraded in application buffer, homogenates of postlarvae prepared in PBS or application buffer and heated at 100 ◦ C for 60 min were analyzed by SDS-PAGE and Western blotting. As a 58 kDa polyhedrin protein band was detected at similar intensity with the application buffer sample compared to the PBS sample (Fig. 2), these data suggested that heating the homogenate in application buffer did not destroy the antigenicity of polyhedrin appreciably, thus limiting MAb binding to polyhedrin protein was caused by reaction between the polyhedrin and the detergent in the application buffer. To minimize time to generate a strip test result, as there was no penalty in band intensity for postlarvae homogenized in PBS heated at 100 ◦ C for 15–60 min, heating for 30 min was selected for further analyses. 3.3. Strip test specificity Only the C band for strip test performance was detected when samples from shrimp infected with WSSV or YHV or TSV or PstDNV were applied to the strip test, indicating that the PemoNPV MAbs used do not cross detect these common shrimp viruses.
Although antibody-based diagnostic methods are generally far less sensitive than PCR-based methods, they offer some advantageous for monitoring viral infections during shrimp cultivation. Among P. monodon farmed in Thailand, individual shrimp has been found commonly to be infected with one or more viruses including PemoNPV, P. monodon densovirus (PmDNV), PstDNV and WSSV, and PemoNPV infections have been detected commonly in shrimp with stunted growth (Flegel et al., 2004). PemoNPV and WSSV have also been detected by PCR in overtly healthy postlarvae being reared in Indian hatcheries (Otta et al., 2003). With WSSV, infection among farmed shrimp can sometimes persist without disease unless triggered by some stress event (Peng et al., 1998; Tsai et al., 1999). PCR does have a clear role in tracking viral infection loads in farmed shrimp to alert looming disease events and to instruct management decisions to minimize the economic impact of disease (Fegan et al., 1991). However, pond-side use of strip tests like that described here should offer to hatchery operators and farmers a simple and quick method for identifying the increases in PemoNPV infection prevalence and loads in their stocks, thus allowing actions to be taken to restrict transmission or limit potential losses due to disease. Acknowledgments We thank the Srinakharinwirot University Research Fund, the Strategic Wisdom and Research Institute of Srinakharinwirot University, for financial support, Pacific Biotech Co. Ltd. for helping produce strip tests and the farmers who provided PemoNPVinfected postlarvae. References
3.4. Strip test sensitivity Using 2-fold dilution series of PemoNPV-infected postlarvae 15 homogenates, positive test-strip results could be scored unambiguously down to a 1:64 detection limit comparable to that obtained in the dot blot method (Fig. 3). In contrast, one-step PCR amplified a 261 bp PemoNPV-specific band with DNA from the same postlarvae homogenate diluted to 10−4 (Fig. 4). While the PemoNPV strip test was ∼200-fold less sensitive than one-step PCR, it fared better than a similar test developed recently to co-detect WSSV and YHV, for which detection sensitivities were ∼500-fold and ∼1000-fold lower, respectively, compared to one-step PCR or RT-PCR tests for these viruses (Sithigorngul et al., 2011). Despite the lower sensitivity of the strip test compared to a one-step PCR test for PemoNPV, it offers simplicity and speed, with results available 15 min after sample preparation, and requires only a basic homogenizer. While offered similar detection sensitivity, dot blots are not amenable to field applications and can take up to 7 h to generate a result.
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Hsu, Y.L., Wang, K.H., Yang, Y.H., Tung, M.C., Hu, C.H., Lo, C.F., Wang, C.H., Hsu, T., 2000. Diagnosis of Penaeus monodon-type baculovirus by PCR and by ELISA of occlusion bodies. Diseases of Aquatic Organisms 40, 93–99. Lu, C.C., Tang, K.F.J., Kou, G.H., Chen, S.N., 1993. Development of a Penaeus monodontype baculovirus (MBV) DNA probe by polymerase chain reaction and sequence analysis. Journal of Fish Diseases 16, 551–559. Nimitphak, T., Meemetta, W., Arunrut, N., Senapin, S., Kiatpathomchai, W., 2010. Rapid and sensitive detection of Penaeus monodon nucleopolyhedrovirus (PemoNPV) by loop-mediated isothermal amplification combined with a lateralflow dipstick. Molecular and Cellular Probes 24, 1–5. Otta, S.K., Karunasaga, I., Karunasaga, I., 2003. Detection of monodon baculovirus and white spot syndrome virus in apparently healthy Penaeus monodon postlarvae from India by polymerase chain reaction. Aquaculture 220, 59–67. Paek, S.H., Lee, S.H., Cho, J.H., Kim, Y.S., 2000. Development of rapid one-step immunochromatographic assay. Methods 22, 53–60. Peng, S.E., Lo, C.F., Liu, K.F., Kou, G.H., 1998. The transition from pre-patent to patent infection of white spot syndrome virus (WSSV) in Penaeus monodon triggered by pereiopod excision. Fish Pathology 33, 395–400. Poulos, B.T., Mari, J., Bonami, J.R., Redman, R., Lightner, D.V., 1994. Used of nonradioactively labeled DNA probes for the detection of a baculovirus from Penaeus monodon by in situ hybridization on fixed tissue. Journal of Virological Methods 49, 187–194. Powell, J.W.B., Borge, E.J., Browdy, C.L., Shepard, E.F., 2006. Efficiency and sensitivity determination of Shrimple® , an immunochromatographic assay for white spot syndrome virus (WSSV), using quantitative real-time PCR. Aquaculture 257, 167–172. Rai, P., Pradeep, B., Karunasagar, I., Karunasagar, I., 2009. Detection of viruses in Penaeus monodon from India showing signs of slow growth syndrome. Aquaculture 289, 231–235. Ramasamy, P., Rajan, P.R., Purushothaman, V., Brennan, G.P., 2000. Ultrastructure and pathogenesis of monodon baculovirus (Pm SNPV) in culture larvae and natural brooders of Penaeus monodon. Aquaculture 184, 45–66. Satidkanitkul, A., Sithigorngul, P., Sang-oum, W., Rukpratanporn, S., Sriurairatana, S., Withyachumnankul, B., Flegel, T.W., 2005. Synthetic peptide used to develop
antibodies for detection of polyhedrin from P. monodon baculovirus (MBV). Diseases of Aquatic Organisms 65, 79–84. Sithigorngul, P., Chauychuwong, P., Sithigorngul, W., Longyant, S., Chaivisuthangkura, P., Menasveta, P., 2000. Development of a monoclonal antibody specific to yellow head virus (YHV) from Penaeus monodon. Diseases of Aquatic Organisms 42, 27–34. Sithigorngul, W., Rukpratanporn, S., Pecharaburanin, N., Longyant, S., Chaivisuthangkura, P., Sithigorngul, P., 2006. A simple and rapid immunochromatographic test strip for detection of white spot syndrome virus (WSSV) of shrimp. Diseases of Aquatic Organisms 72, 101–106. Sithigorngul, W., Rukpratanporn, S., Sittidilokratna, N., Pecharaburanin, N., Longyant, S., Chaivisuthangkura, P., Sithigorngul, P., 2007. A convenient immunochromatographic test strip for rapid diagnosis of yellow head virus infection in shrimp. Journal of Virological Methods 140, 193–199. Sithigorngul, P., Rukpratanporn, S., Chaivisuthangkura, P., Sridulyakul, P., Longyant, S., 2011. Simultaneous and rapid detection of white spot syndrome virus and yellow head virus infection in shrimp with a dual immunochromatographic strip test. Journal of Virological Methods 173, 85–91. Sridulyakul, P., Suwannaka, T., Chaivisuthangkura, P., Longyant, S., Rukpratanporn, S., Sithigorngul, P., 2011. Penaeus monodon nucleopolyhedrosisvirus detection using monoclonal antibodies specific to recombinant polyhedrin protein. Aquaculture 321, 216–222. Surachetpong, W., Poulos, B.T., Tang, K.F.J., Lightner, D.V., 2005. Improvement of PCR method for detection of monodon baculovirus (MBV) in penaeid shrimp. Aquaculture 249, 69–75. Tsai, M.F., Kuo, G.H., Liu, H.C., Liu, K.F., Chang, C.F., Peng, S.E., Hsu, H.C., Wang, C.H., Lo, C.F., 1999. Long-term presence of white spot syndrome virus (WSSV) in a cultivated shrimp population without disease outbreaks. Diseases of Aquatic Organisms 38, 107–114. Vaseeharan, B., Ramasamy, P., 2003. Abundance of potentially pathogenic microorganisms in Penaeus monodon larvae rearing systems in India. Microbiological Research 158, 299–308. Yan, D., Tang, K.F.J., Lightner, D.V., 2009. Development of a real-time PCR assay for detection of monodon baculovirus (MBV) in penaeid shrimp. Journal of Invertebrate Pathology 102, 97–100.
Contents lists available at SciVerse ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Penaeus monodon nucleopolyhedrovirus detection using an immunochromatographic strip test Pradit Wangman a , Siwaporn Longyant a , Parin Chaivisuthangkura a , Pattarin Sridulyakul a , Sombat Rukpratanporn b , Paisarn Sithigorngul a,∗ a b
Department of Biology, Srinakharinwirot University, Bangkok 10110, Thailand Center of Excellence for Marine Biotechnology at Chulalongkorn University, National Center for Genetic Engineering and Biotechnology (BIOTEC), Bangkok 10330, Thailand
a b s t r a c t Article history: Received 8 December 2011 Received in revised form 3 April 2012 Accepted 30 April 2012 Available online 10 May 2012 Keywords: Immunochromatographic strip test Monoclonal antibody Polyhedrin Penaeus monodon nucleopolyhedrovirus (PemoNPV) Monodon baculovirus (MBV)
An immunochromatographic strip test is described for detection of the polyhedrin protein of Penaeus monodon nucleopolyhedrovirus (PemoNPV). The test employs one monoclonal antibody (MAb MBV5) conjugated to colloidal gold to bind to polyhedrin protein and a 1:1:1 mixture of 3 other MAbs (MBV8, 14 and 21) to capture colloidal-gold MAb–protein complexes at a test (T) line on the nitrocellulose strip. A downstream control (C) line of goat anti-mouse immunoglobulin G (GAM) antibody is used to capture excess free colloidal-gold conjugated MBV5 to validate test performance. Heating of homogenates of PemoNPV-infected P. monodon postlarvae prepared in PBS for 30 min was necessary to maximize T line color intensity, and homogenates of infected postlarvae could still be scored as PemoNPV-positive when diluted 1:64. A strip test result was obtained within 15 min of sample application, and although about 200-fold lower than a one-step PCR test for PemoNPV, its detection sensitivity was comparable to a dot blot. Due to its simplicity not reliant on sophisticated equipment or specialized skills, the strip test could be adopted to screen easily for PemoNPV infections at shrimp hatcheries and farms. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Penaeus monodon nucleopolyhedrovirus (PemoNPV) or monodon baculovirus (MBV) as it has been called previously, occurs commonly as a subclinical infection in black tiger (P. monodon) shrimp broodstock but can cause disease in early life stages (larvae, protozoea, mysis, postlarvae) or compromise their resilience against Vibrio spp. and other protozoa (Ramasamy et al., 2000; Vaseeharan and Ramasamy, 2003). Although PemoNPV infection is not associated generally with mortalities in farmed P. monodon (Fegan et al., 1991), it has been linked circumstantially with stunting (Flegel et al., 1999, 2004; Rai et al., 2009). PemoNPV infection can be diagnosed using squash mounts of shrimp hepatopancreas tissue and malachite green to stain intranuclear occlusion bodies or using histology to identify cells with enlarged nuclei containing acidophilic occlusions (Flegel, 2006). Polymerase chain reaction (PCR) tests (Chang et al., 1993; Lu et al., 1993; Belcher and Young, 1998; Hsu et al., 2000) and in situ hybridization assays on histological sections are also available to detect PemoNPV (Poulos et al., 1994). Although the PCR method described by Belcher and Young (1998) proved more reliable for detecting PemoNPV strains in shrimp from Australia, India and
Thailand (Flegel, 2006), a revised PCR test has since been proven to be useful for detecting strains from ten geographically disparate regions (Surachetpong et al., 2005). Real-time PCR (Yan et al., 2009) and loop-mediated isothermal amplification (LAMP) methods (Chaivisuthangkura et al., 2009; Nimitphak et al., 2010) have also been shown to be effective in detecting PemoNPV strains from Thailand and Indonesia. Methods using polyclonal or monoclonal antibodies (MAbs) have been developed for the detection of several shrimp pathogens including PemoNPV (Satidkanitkul et al., 2005; Boonsanongchokying et al., 2006; Sridulyakul et al., 2011). Although less sensitive than PCR, such antibody-based diagnostic methods tend to be simpler, less expensive and more amenable to use in less well equipped laboratories or by unskilled personnel in the field (Powell et al., 2006; Sithigorngul et al., 2006, 2007, 2011). Here, a simple immunochromatographic strip test method able to detect rapidly PemoNPV in P. monodon postlarvae is described that employs a set of four MAbs specific to the PemoNPV polyhedrin protein (Boonsanongchokying et al., 2006).
2. Materials and methods 2.1. Virus
∗ Corresponding author. Tel.: +66 2 664 1000x8515; fax: +66 2 260 0127. E-mail addresses: paisarn [email protected], [email protected] (P. Sithigorngul). 0166-0934/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2012.04.016
PemoNPV-infection in P. monodon postlarvae 10 and postlarvae 15 collected from a farm in Chonburi province, Thailand
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was verified using one-step PCR (Surachetpong et al., 2005). Individual postlarvae were homogenized in 50 l PBS (0.15 M phosphate buffered saline, pH 7.2), heated at 100 ◦ C for 30 min and homogenate aliquots were stored at −20 ◦ C. 2.2. Monoclonal antibody preparation The four monoclonal antibodies (MAbs MBV5, 8, 14, and 21) used in the strip test are reactive to different epitopes on the PemoNPV polyhedrin protein and were produced as described previously from a mouse immunized with the native protein (Boonsanongchokying et al., 2006). MAbs secreted from hybridoma cells grown in serum-free Hybridoma-SFM media (Gibco, Grand Island, NY, USA) were purified using Protein G-agarose columns (Roche Molecular Biochemicals, Indianapolis, IN, USA) according to the manufacturer’s instructions. The MAbs were dialyzed against 10 mM phosphate buffer, pH 7.3 and adjusted to a concentration of 1 mg/ml before being stored in aliquots at −20 ◦ C. 2.3. Dot blot Aliquots of 2-fold dilution series of homogenates of uninfected postlarvae 10 or PemoNPV-infected postlarvae 15 were spotted (1 l/spot) onto nitrocellulose membranes and baked at 60 ◦ C for 10 min. After incubating for 4 h in various hybridoma culture media diluted 1:20 in 5% blocking solution (5% nonfat dry milk and 0.1% Triton X-100 in PBS), bound MAbs were detected by incubation for 3 h in 1:1500 horseradish peroxidase-labeled goat anti-mouse gamma immunoglobulin heavy and light chain-specific antibody (GAM-HRP, Bio-Rad, Hercules, CA, USA) followed by incubation in 0.03% diaminobenzidine (DAB) substrate in 0.006% H2 O2 , 0.05% cobalt chloride in PBS as described previously (Sithigorngul et al., 2000). 2.4. Immunochromatographic test strip The layout, assembly and operation of the immunochromatographic test strip based on 8 m pore-size nitrocellulose (NC) membranes (Pacific Biotech Co. Ltd., Thailand) have been described in detail previously (Sithigorngul et al., 2011). Briefly, the MAb MBV5 used to capture PemoNPV polyhedrin protein from samples was conjugated to 10 nm diameter colloidal gold particles and sprayed (3 l/cm) onto a glass fiber pad and dried overnight at 40 ◦ C. On the NC membrane, a 1:1:1 mixture of 1 mg/ml MAbs MBV8, MBV14 and MBV21 was micro-sprayed (1 l/cm) at the test (T) line position and goat anti-mouse IgG antibody (0.8 mg/ml) was micro-sprayed (1 l/cm) at the control (C) line position, and the membranes were then dried overnight at 40 ◦ C. The pad containing gold-conjugated MBV5 was placed between the well for sample application and the T line, and an absorption pad was placed downstream of the C line. The assembled test strips were stored desiccated in a plastic bag until used. 2.5. Optimizing sample preparation for strip test analysis As PemoNPV polyhedrin protein aggregate to form dense occlusion bodies in the nucleus, antibody access is limited to the occlusion body surface. To optimize occlusion body disruption, homogenates of individual postlarvae 15 diluted in 50 l PBS or application buffer (30 mM Tris, 336 mM NaCl, 9 nM EDTA, 1% Triton X-100, pH 9.3) were heated at 100 ◦ C for various time periods (0, 5, 15, 30 and 60 min). After mixing with an equal volume of application buffer, 100 l homogenate was applied to the strip test. To identify whether negative detections with postlarvae homogenized and heated in application buffer were not due to the polyhedrin protein degradation, homogenates diluted in PBS
Fig. 1. PemoNPV strip test results using homogenates of (A) PemoNPV-infected P. monodon postlarvae 15 and (B) uninfected postlarvae 10. (For interpretation of the references to color in the text, the reader is referred to the web version of the article.)
or application buffer and heated for 60 min were subjected to SDS-PAGE and Western blot analysis as described previously (Sridulyakul et al., 2011) using MAb MBV8. 2.6. Specificity testing Individual uninfected or PemoNPV-infected P. monodon postlarvae 8, postlarvae 10 and postlarvae 15 were homogenized in 50 l PBS, boiled for 30 min, mixed 1:1 with application buffer and analyzed in the strip test. Reddish-purple colored bands developed within 15 min at both T and C lines when PemoNPV was detected but only at the C line when it was not detected (Fig. 1). To assess strip test specificity against common shrimp viruses, homogenates of pleopods from shrimp infected with white spot syndrome virus (WSSV), yellow head virus (YHV), Taura syndrome virus (TSV) or Penaeus stylirostris densovirus (PstDNV) were applied. 2.7. Strip test sensitivity compared to dot blot and PCR Individual uninfected P. monodon postlarvae 10 or PemoNPVinfected postlarvae 15 were homogenized in 50 l PBS and heated at 100 ◦ C for 30 min. Aliquots (1 l) of this homogenate were used directly to prepare dilution series for dot blot analysis or mixed 1:1 in application buffer before analysis using the strip test or and PCR. Nucleic acid extracted from 100 l homogenate using a High Pure Viral Nucleic Acid Kit (Roche Molecular Biochemicals) and diluted serially in 50 ng/l nucleic acid extracted from uninfected shrimp was tested using a PemoNPV PCR method described previously (Surachetpong et al., 2005). Aliquots (100 l) of these serial dilutions were also applied to immunochromatographic test strips. 2.8. Thermostability of the strip test To assess thermostability, immunochromatographic test strips in a desiccated plastic bag were placed in a 60 ◦ C oven for 10, 20 or 30 days before use with homogenates of uninfected or PemoNPVinfected postlarvae diluted 1:5 or 1:50 in application buffer (10 strips tested per time point). 3. Results and discussion 3.1. Strip test optimization In the optimized strip test method, four MAbs targeted to different epitopes were combined with different combinations and used for colloidal gold conjugate or test line. Finally MAbs were selected for gold-labeling and initial binding (MBV5) and
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Fig. 2. Optimization of sample preparation methods for the PemoNPV strip test. Postlarvae homogenized in (A) PBS or (B) application buffer were heated at 100 ◦ C for 0, 5, 15, 30 and 60 min. Postlarvae homogenized in PBS and application buffer and heated for 60 min (arrowheads) were also subjected to SDS-PAGE and Western blot analysis to detect PemoNPV polyhedrin protein (58 kDa). (For interpretation of the references to color in the text, the reader is referred to the web version of the article.)
capture of PemoNPV polyhedrin protein (MAbs MBV8, MBV14, and MBV21) based of this combination resulting in the highest band color intensity and sensitivity based on detection of serial dilutions of PemoNPV-infected P. monodon postlarvae homogenates without giving false-positives with homogenates prepared from uninfected postlarvae (Fig. 1).
3.2. Sample preparation method Among individual PemoNPV-infected postlarvae homogenized in PBS, no band resulted without heating, heating at 100 ◦ C for 5 min resulted in a lightly reddish purple colored band and heating for 15, 30 or 60 min resulted in darkly reddish purple colored bands
Fig. 3. Sensitivity limit of the strip test and dot blots for detecting PemoNPV. (A) Strip test detection of serial 2-fold dilutions of PemoNPV-infected postlarvae 15 homogenized in PBS, (B) dot blot detection of serial 2-fold dilutions of postlarvae homogenate using each single MAb or all 4 MAbs combined. N = uninfected postlarvae homogenate. Arrowheads indicate the lowest amount detected reliably.
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3.5. Strip test thermostability Using strip tests stored desiccated in a 60 ◦ C oven for various time periods, PemoNPV-infected postlarvae homogenates diluted 1:5 were detected equally well with strips stored for up to 20 days, but slightly less well using strips stored for 30 days (data not shown). With PemoNPV-infected postlarvae homogenates diluted 1:50, the color intensity of positive bands was comparable with strips stored at 60 ◦ C for up to 30 days. Data obtained with strips heated at this temperature indicate that the strip tests should remain functional for up to 2 years when stored at room temperature (Paek et al., 2000), as found similarly for the strip tests for WSSV and YHV (Sithigorngul et al., 2006, 2007, 2011). 4. Conclusions Fig. 4. One-step PCR detection of PemoNPV DNA in nucleic acid extracted from PemoNPV-infected postlarvae 15 from Fig. 3 and serially diluted 10-fold in nucleic acid from an uninfected postlarvae. Lane M: DNA markers; Lanes (a) 10−1 , (b) 10−2 , (c) 10−3 , (d) 10−4 , (e) 10−5 dilutions, and (f) uninfected postlarvae homogenate. The arrowhead indicates the detection limit of the 261 bp PCR product.
similar in intensity (Fig. 2A). Among postlarvae homogenized in application buffer, no band also resulted without heating, and heating for any length of time only resulted in a lightly gray colored band (Fig. 2B) of non-specific reaction. To confirm that the polyhedrin protein had not degraded in application buffer, homogenates of postlarvae prepared in PBS or application buffer and heated at 100 ◦ C for 60 min were analyzed by SDS-PAGE and Western blotting. As a 58 kDa polyhedrin protein band was detected at similar intensity with the application buffer sample compared to the PBS sample (Fig. 2), these data suggested that heating the homogenate in application buffer did not destroy the antigenicity of polyhedrin appreciably, thus limiting MAb binding to polyhedrin protein was caused by reaction between the polyhedrin and the detergent in the application buffer. To minimize time to generate a strip test result, as there was no penalty in band intensity for postlarvae homogenized in PBS heated at 100 ◦ C for 15–60 min, heating for 30 min was selected for further analyses. 3.3. Strip test specificity Only the C band for strip test performance was detected when samples from shrimp infected with WSSV or YHV or TSV or PstDNV were applied to the strip test, indicating that the PemoNPV MAbs used do not cross detect these common shrimp viruses.
Although antibody-based diagnostic methods are generally far less sensitive than PCR-based methods, they offer some advantageous for monitoring viral infections during shrimp cultivation. Among P. monodon farmed in Thailand, individual shrimp has been found commonly to be infected with one or more viruses including PemoNPV, P. monodon densovirus (PmDNV), PstDNV and WSSV, and PemoNPV infections have been detected commonly in shrimp with stunted growth (Flegel et al., 2004). PemoNPV and WSSV have also been detected by PCR in overtly healthy postlarvae being reared in Indian hatcheries (Otta et al., 2003). With WSSV, infection among farmed shrimp can sometimes persist without disease unless triggered by some stress event (Peng et al., 1998; Tsai et al., 1999). PCR does have a clear role in tracking viral infection loads in farmed shrimp to alert looming disease events and to instruct management decisions to minimize the economic impact of disease (Fegan et al., 1991). However, pond-side use of strip tests like that described here should offer to hatchery operators and farmers a simple and quick method for identifying the increases in PemoNPV infection prevalence and loads in their stocks, thus allowing actions to be taken to restrict transmission or limit potential losses due to disease. Acknowledgments We thank the Srinakharinwirot University Research Fund, the Strategic Wisdom and Research Institute of Srinakharinwirot University, for financial support, Pacific Biotech Co. Ltd. for helping produce strip tests and the farmers who provided PemoNPVinfected postlarvae. References
3.4. Strip test sensitivity Using 2-fold dilution series of PemoNPV-infected postlarvae 15 homogenates, positive test-strip results could be scored unambiguously down to a 1:64 detection limit comparable to that obtained in the dot blot method (Fig. 3). In contrast, one-step PCR amplified a 261 bp PemoNPV-specific band with DNA from the same postlarvae homogenate diluted to 10−4 (Fig. 4). While the PemoNPV strip test was ∼200-fold less sensitive than one-step PCR, it fared better than a similar test developed recently to co-detect WSSV and YHV, for which detection sensitivities were ∼500-fold and ∼1000-fold lower, respectively, compared to one-step PCR or RT-PCR tests for these viruses (Sithigorngul et al., 2011). Despite the lower sensitivity of the strip test compared to a one-step PCR test for PemoNPV, it offers simplicity and speed, with results available 15 min after sample preparation, and requires only a basic homogenizer. While offered similar detection sensitivity, dot blots are not amenable to field applications and can take up to 7 h to generate a result.
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