Simultaneous PCR detection of two shrimp viruses (WSSV and MBV) in postlarvae of Penaeus monodon in the Philippines

Aquaculture 257 (2006) 142 – 149 www.elsevier.com/locate/aqua-online

Simultaneous PCR detection of two shrimp viruses (WSSV and MBV) in postlarvae of Penaeus monodon in the Philippines Karlo Dante T. Natividad a,c , Maria Veron P. Migo b , Juan D. Albaladejo b , Jose Paolo V. Magbanua c , Nakao Nomura a , Masatoshi Matsumura a,⁎ a

c

Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennodai 1-1-1, Tsukuba, 305-8572, Japan b Bureau of Fisheries and Aquatic Resources (BFAR), Department of Agriculture, Quezon City 1103, Philippines National Institute of Molecular Biology and Biotechnology (BIOTECH), University of the Philippines-Los Baños, College, Laguna 4031, Philippines Received 5 December 2005; received in revised form 27 February 2006; accepted 28 February 2006

Abstract A duplex polymerase chain reaction (PCR) protocol was developed for the simultaneous detection of two penaeid shrimp viruses, namely, White spot syndrome virus (WSSV) and Monodon baculovirus (MBV) infecting Penaeus monodon in Philippines. The method was designed for screening postlarval samples with dual infections of MBV and WSSV. The developed protocol was able to generate a 211 bp amplicon which is highly specific for WSSV and a 361 bp amplicon specific for MBV. An assessment of the sensitivity of the developed duplex PCR demonstrated the detection of both the amplicons up to 0.1 femtogram (fg) of plasmid DNA containing the target sequences equivalent to 15 copies of the viral target sequence. In addition to its high specificity and sensitivity, the developed duplex PCR offers an efficient and rapid tool for screening penaeid shrimp viruses since both WSSV and MBV can be diagnosed in a single reaction. © 2006 Elsevier B.V. All rights reserved. Keywords: White spot syndrome virus; Monodon baculovirus; Duplex PCR

1. Introduction Shrimp aquaculture used to be a lucrative industry until disease outbreaks wreaked havoc worldwide, especially in Asian countries, including the Philippines. Among the shrimp pathogens, viruses have undoubtedly caused the most severe losses to shrimp farmers (Flegel, 2002). To date, more than 20 penaeid shrimp viruses Abbreviations: WSSV, White spot syndrome virus; MBV, Monodon baculovirus; PL, Postlarvae. ⁎ Corresponding author. Institute of Applied Biochemistry, University of Tsukuba, Tennodai 1-1-1, Tsukuba City, 305-8572, Japan. Tel./ fax: +81 29 853 6624. E-mail address: [email protected] (M. Matsumura) 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.02.061

were identified as having affected wild stocks and commercial production (Hernández-Rodríguez et al., 2001). White spot syndrome virus (WSSV), first reported in 1993, is one of the most widespread and devastating infectious agents that has hit the shrimp aquaculture industry. The disease develops rapidly reaching 100% cumulative mortality within 3–10 days post-infection in experimentally infected animals. Intensive cultivation, inadequate sanitation and worldwide uncontrolled stock movements have quickly spread the disease to major shrimp farming areas (Rosenberry, 2000). However, it was only in 1999 that the first incidence of WSSV in Philippines was reported (Magbanua et al., 2000). In an

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effort to contain the spread of the infectious agent in farms and hatcheries, the Philippine government through the Bureau of Fisheries and Aquatic Resources (BFAR) has modified routine fry quality assessment to include screening for WSSV. For definitive diagnosis and certification of WSSV infection status of broodstock and fry, polymerase chain reaction (PCR) technology is recommended. However, definitive diagnosis can also be accomplished by in situ DNA hybridisation, Western blot analysis and transmission electron microscopy (TEM) (OIE, 2003). Another shrimp pathogen analyzed for fry quality assessment is monodon baculovirus (MBV). Although not as lethal as WSSV, MBV is considered to be a potentially serious pathogen in the larval, postlarval and juvenile stages of penaied shrimp. MBV infections are characterised by the presence of prominent, spherical intranuclear occlusion bodies in affected epithelial cells of the hepatopancreas and midgut, or free within lysed cell debris in the faeces (Lightner, 1996; BondadReantaso et al., 2001). Although good culture practices may enhance the survival of MBV-infected stocks, growth, crop value and performance may significantly be reduced and MBV may render the infected shrimp susceptible to other pathogens with higher mortality rates (Bower, 1996). Diagnosis of MBV infection is usually by microscopic examination of characteristic occlusion bodies produced by the virus. However, molecular methods for MBV surveillance are also available. These include an antibody-based enzyme linked immunosorbent assay (ELISA) (Hsu et al., 2000), gene probes for in situ hybridization (Lightner and Redman, 1998) and DNA-based PCR protocols (Vickers et al., 1992; Belcher and Young, 1998). Dual infections of MBV and WSSV have been observed in wild black tiger shrimp from several sampling sites in the Philippines using PCR (De la Pena et al., 2005). In similar cases, mixed infections of WSSV and MBV were also reported in other Asian countries like Vietnam (Hao et al., 1999), Thailand (Flegel et al., 2004) and India wherein 18% of the total samples analyzed showed dual infection of MBV and WSSV in apparently healthy PL using Nested PCR (Otta et al., 2003).

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Since WSSV and MBV analysis is routine for fry quality assessment in the Philippines to lessen the risk of disease outbreaks, this paper describes a DNA-based duplex PCR for the simultaneous detection of both viruses in P. monodon postlarvae with dual infections. With the developed duplex PCR, detection and differentiation of both viruses could be achieved in a single reaction. Thus would greatly improve handling efficiency for rapid detection of these shrimp pathogens. 2. Materials and methods 2.1. Sample preparation and DNA isolation Approximately 50–100 live P. monodon postlarvae (PL) were homogenized in 10% (w/v) Tris–NaCl– EDTA (TNE) buffer using a sterile mortar and pestle. The homogenate was centrifuged at 3000 ×g for 15 min. A 100-μl aliquot of the supernatant was mixed with 500 μl DNAZol Reagent (Invitrogen), and DNA was extracted according to the manufacturers' protocol. 2.2. Polymerase chain reaction (PCR) Samples were analyzed for the presence of WSSV and MBV using polymerase chain reaction. Primers used for the amplification of WSSV and MBV are listed in Table 1. For convenience, the forward and reverse primers for WSSV described by Tapay et al. (1999) were designated as LMT1 and LMT2 respectively. A single PCR reaction mixture containing 1× PCR buffer with MgCl2, 200 μM dNTP mix, 1.25 U of Ex Taq polymerase (TaKaRa Bio Inc, Japan), 0.4 μM of forward and reverse primers and 5 μl template DNA was prepared. All PCR experiments were carried out in 0.2 ml tubes in a BioRad thermal cycler with the following cycle parameters: intitial denaturation at 95 °C for 5 min, followed by 30 cycles of 95 °C for 30 s, annealing temp 60 °C (WSSV) and 65 °C (MBV) for 1 min and 72 °C for 1 min, and a final extension of 72 °C for 5 min. PCR products were analyzed in agarose gel (1.5% in Tris-acetate EDTA buffer) containing 0.5 μg/ml ethidium bromide. A 100 bp DNA ladder was used as molecular weight (MW) marker. Electrophoresis

Table 1 Details of primers used for the amplification of WSSV and MBV gene by PCR Virus

Primer name

Primer sequence (5′–3′)

Tm (°C)

Size (bp)

Reference

WSSV

LMT1 LMT2 MBV1.4NF MBV1.4NR

5′-GAA ACT ATT GAA AAG GCT TTC CCT C-3′ 5′-GTT CCT TAT TTA CTA CTA CGG CAA-3′ 5′-TCC AAT CGC GTC TGC GAT ACT-3′ 5′-CGC TAA TGG GGC ACA AGT CTC-3′

57.2 55.3 58.5 60.4

211

Tapay et al., 1999

361

Belcher and Young, 1998

MBV

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was carried out at 100 V in a Mupid 21 mini-gel electrophoresis unit for 30 min and viewed under a UV transilluminator. 2.3. Duplex PCR protocol The same targets as those for the individual PCRs were used for the single reaction Duplex PCR. Two primer pairs were used, namely, LMT1, LMT2 and MBV1.NF, MBV1.NR (Table 1). Because of the difference in melting temperature (Tm) of the primers, optimum annealing temperature that would amplify both targets was determined using BioRad iCycler Gradient PCR. A reaction mixture containing 1× PCR buffer with MgCl2, 250 μM dNTP mix, 1.25 U of Ex Taq polymerase (TaKaRa Bio Inc, Japan), 0.4 μM of each LMT1, LMT2, MBV1.NF and MBV1.NR primers was prepared. Template DNA used was an equal volume mixture of DNA from previously identified WSSV and MBV positive samples. Cycle parameters are as follows: initial denaturation at 95 °C for 5 min, followed by 35 cycles of 95 °C for 30 s, annealing temp 60 °C for 1 min and 72 °C for 1 min, and a final extension of 72 °C for 5 min. 2.4. Cloning and sequencing of amplified WSSV and MBV gene Amplified PCR products were purified using a gel extraction kit (Qiagen). Purified MBV and WSSV DNA fragments were ligated in pET 100 (Invitrogen) and transformed in E. coli TOP10 (Invitrogen). Plasmids containing the insert (designated as pWSSV and pMBV, for WSSV and MBV inserts, respectively) were extracted using Purelink HQ Mini-Plasmid Purification Kit (Invitrogen). Plasmid DNA concentration was determined by taking the absorbance at 260 nm using an Ultrospec 3300 Pro (Amersham Biosciences) and purity was confirmed using the 260/280 nm ratio. Presence of the insert was confirmed by PCR amplification using the plasmid as template. DNA sequence was determined using the dideoxy-chain termination method (Sanger et al., 1977) with a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and ABI PRISM™ 310 Genetic Analyzer (Perkin Elmer). Homology analysis of the sequence results was done by BLAST search at the National Center for Biotechnology Information (NCBI) database. 2.5. Sensitivity analysis of the duplex PCR The sensitivity of the duplex PCR was determined using different concentrations of template DNA.

Template DNA was prepared as follows: equal concentrations of the plasmids containing the PCR target sequences for WSSV and MBV were mixed and serially diluted in ten-fold steps from 100 ng/μl to 0.01 fg/μl using sterile distilled water. Two microliters from each dilution was used as template and subjected to the duplex PCR protocol. The limit of detection of the duplex PCR was determined based on the highest dilution that resulted in the presence of both WSSV and MBV amplified fragments on the agarose gel. 3. Results 3.1. Primer selection and specificity Primer pairs LMT1 and LMT2 (Tapay et al., 1999) and MBV1.4NF and MBV1.4NR (Belcher and Young, 1998) were selected because of their successful amplification of WSSV and MBV DNA, respectively. Initially, both primers were evaluated for the amplification of Philippine strains of WSSV and MBV. For the amplification of WSSV, three primer pairs were compared, namely LMT1 and LMT2, PRDVF and PRDVR (Takahashi et al., 1996), 146F1 and 146R1 (Lo et al., 1996). Templates used for the amplification were gills from WSSV-infected P. monodon and P. japonicus. Results showed that LMT1 and LMT2 primers consistently amplified target sequences from all tested Philippine and Japanese WSSV isolates. PRDVF and PRDVR primers amplified target sequences from one WSSV isolate from the Philippines and one from Japan, while 146F and 146R primers were able to amplify only the isolate from Japan (Fig. 1). Based on the results and its consistency, LMT primer pair was chosen and used in the succeeding experiments. MBV1.4NF and MBV1.4NR were first evaluated for the amplification of MBV from P. monodon postlarval samples obtained from various hatcheries in the Philippines. PCR results for the presence of MBV coincided with the results of direct microscopic examination (data not shown), thus suggesting that these primers are suitable for the detection of Philippine strain of MBV. Specificity of both primers was reassessed using Philippine strains of MBV and WSSV. DNA from WSSV- and MBV-infected P. monodon postlarvae (PL) were used as templates. Uninfected (healthy) P. monodon PL served as negative control. MBV1.4NF and MBV1.4NR yielded the expected 361 bp amplicon from MBV-infected PL, and not from WSSV-infected PL DNA (Fig. 2). On the other hand, LMT1 and LMT2 primers amplified the expected 211 bp amplicon from

K.D.T. Natividad et al. / Aquaculture 257 (2006) 142–149 LMT primer

1

2

3

4

146 primer

5

6

7

8

145

PRDV primer

9

10

11 12 13

MW= bp

1447 bp

643 bp 500 211 bp 200

211 bp

Fig. 1. WSSV analysis using different sets of primers. Lanes 2, 3, 6, 7, 10 and 11, DNA template from WSSV-infected P. monodon; lanes 4, 8 and 12, DNA template from WSSV-infected P. japonicus; lanes 5, 9 and 13, DNA template from healthy P. monodon (negative control).

WSSV-infected PL only. More importantly, DNA from healthy PL did not yield any PCR product, suggesting that these primers were specific to WSSV and MBV. Both primers used have been widely adopted for WSSV and MBV detection and no cross reactions with other shrimp viruses have ever been reported. Specificity of both primers was successfully established in order to avoid false positive results especially when dealing with PLs with mixed infections of WSSV and MBV and healthy PLs. 3.2. Sequence analysis of the amplified fragments To confirm whether the amplified fragments were indeed WSSVor MBV DNA and not from PL DNA, the M BV Primer

MW =bp

1

2

3

4

WSSV Primer

5

6

7

500 361 bp b 200

211 bp b

fragments were cloned and sequenced. Sequence analysis of the 211-bp amplicon from WSSV-infected PL revealed a 99% homology to a single copy representing a fraction of the ORF119 (Gene family 8) of the WSSV genome (AY864671). It also shared a 99% homology with the sequence previously reported by Tapay et al. (1999). Sequence results suggest that the primers used were able to amplify highly conserved sequence among different geographical isolates of WSSV. The high homology of the target sequence also indicates that the WSSV Philippine strain is closely related to the published WSSV genome of the Thailand isolate (van Hulten et al., 2001). On the other hand, sequence analysis of the MBV 361 amplicon revealed a 92.7% homology with the sequence published by Belcher and Young (1998). Other MBV isolates with reported homology with the sequence by the said authors were from Taiwan (91.8%), Thailand (92.3%), Indonesia (92.1%), and India (94.1%) (Farming IntelliGene Technology Corporation Web page). NCBI BLAST results of the 361 bp amplicon also showed a 95% homology with MBV-TN2 strain (AY494592) from India. Variation in nucleic acid sequence of the 361 amplicon may be attributed to the wide geographical distribution and diverse host range of MBV. Thus, multiple strains of the virus are likely to exist (Natividad and Lightner, 1992). 3.3. Duplex PCR: optimum annealing temperature

Fig. 2. PCR results showing the specificity of the primers used for duplex PCR protocol. Lane 1, 100 bp MW marker; lane 2, MBVinfected PL; lane 3, WSSV-infected PL; lane 4, healthy PL; lane 5, WSSV-infected PL; lane 6, MBV-infected PL; lane 7, healthy PL. Lanes 2–4 were amplified using MBV primers and lanes 5–7 were amplified using WSSV primers.

The optimum annealing temperature that would yield similar amounts of both amplicons (211 and 361 bp) was determined to minimize the bias caused by the difference in melting temperature (Tm) of the MBV and

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1

2

3

4

5

6

7

8

9

MW= bp

500 361 bp 200

211 bp

Fig. 3. Effect of various annealing temperatures (°C) on the amount of PCR product. Lane 1, 100 bp MW marker; lane 2, 65 °C ; lane 3, 64 °C ; lane 4, 62 °C ; lane 5, 60 °C; lane 6, 56 °C ; lane 7, 53 °C ; lane 8, 51 °C ; lane 9, 50 °C. Arrowheads showing the 211 bp WSSV amplicon and the 361 bp MBV amplicon.

WSSV primers. Duplex PCR reactions were run at various annealing temperatures (50, 51, 53, 56, 60, 62, 64 and 65 °C). Results showed that at high annealing temperatures (N 60 °C) only the 361 bp amplicon was generated while at low annealing temperatures (b 58 °C) only the 211 bp amplicon was generated (Fig. 3). Based on this data, an annealing temperature of 60 °C was optimum for the duplex PCR since similar amounts of both amplicons were generated. 3.4. Duplex PCR: specificity The specificity of the duplex-PCR protocol was evaluated using naturally infected samples. Mixed DNA from MBV- and WSSV-infected PL and DNA from healthy PL were used as positive and negative controls, respectively. The duplex PCR protocol exhibited specificity towards its target sequences since no product was generated when using DNA from healthy PL as template. Only the 211 bp amplicon was generated when using DNA from WSSV-infected PL and only the 361 bp amplicon when DNA from MBV-infected PL was used as template (Fig. 4). The method was initially tested in samples found to be infected with MBV based on direct microscopic examination. Using our duplex PCR, two samples were found positive for both MBV and WSSV as both bands were present in the gel; although the severity of WSSV infection was considered light as the 211-bp WSSV band was less intense as compared to the MBV band (Fig. 4, lane 6). 3.5. Duplex PCR: sensitivity To determine the sensitivity of the duplex PCR protocol, equal concentrations of pWSSV (ca. 6.0 kb)

and pMBV (ca. 6.1 kb) were mixed and diluted in tenfold steps from 100 ng/μl to 0.01 fg/μl. From each dilution, 2 μl were used as DNA template for the duplex PCR. Taking into account the size of the plasmid vector and the insert, 1 fg of mixed DNA contains approximately 75 copies of each of plasmid insert. Both WSSV and MBV bands were visible from 100 ng to 0.1 fg DNA (Fig. 5). Moreover, the WSSV band was still visible even in 0.01 fg of DNA, but not the MBV band. However, it is possible that the MBV band is amplified, but the amount is less than the detection limit of the ethidium bromide stain. This suggests that the detection limit of the developed duplex PCR was 0.1 fg of plasmid DNA containing 15 copies of target sequences (or 15 viral genome equivalents). It should be noted that this estimate on the viral genome equivalent is based on the

1

2

3

4

5

6

MW= bp

500 361 bp 200

211 bp

Fig. 4. Duplex PCR results showing the 361 bp MBV amplicon and 211 bp WSSV amplicon. Lane 1, 100 bp MW Marker; lane 2, mixed DNA from MBV-infected and WSSV-infected samples (positive control); lane 3, DNA from healthy PL (negative control); lane 4, DNA from WSSV-infected PL; lane 5, DNA from MBV-infected PL; lane 6, DNA from PL with mixed infections of WSSV and MBV.

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147

1 ng

100 pg

10 pg

1 pg

100 fg

10 fg

3

4

5

6

7

8

9

10

0.01 fg

10 ng

2

0.1 fg

100 ng

1

1 fg

Marker

Plasmid DNA Concentration

11 12

MW= bp

500 200

361 bp 211 bp

Fig. 5. Duplex PCR sensitivity analysis using plasmid DNA containing the target sequences as PCR template. Plasmid DNA was serially diluted from 100 ng to 0.01 fg (lanes 2–12). Lane 1, 100 bp MW marker. Bottom right arrowhead shows the position of excess primers of the reaction.

assumption that there is only one copy of the target sequence per viral genome. 4. Discussion Among the penaeid shrimp viruses, MBV and WSSV are widespread and are currently under surveillance in the Philippines. In 1992, Natividad and Lightner reported an 85–100% prevalence of MBV in P. monodon postlarvae in the Philippines, while in 2004 there was a 59% prevalence of MBV in P. monodon postlarvae based on the fry quality assessment results from the Bureau of Fisheries and Aquatic Resources (BFAR). On the other hand, WSSV was first reported in the Philippines in 1999, wherein 72% of the samples analyzed were positive for WSSV (Magbanua et al., 2000). Last year alone (2004), 303 samples were analyzed and 58 (19%) were positive for WSSV. Most of the samples that were positive for WSSV were obtained from farms experiencing mortalities due to unknown causes. Due to higher sensitivity limits than most classical diagnostic methods, PCR has become the preferred method for diagnosis of most shrimp viruses (Tang and Lightner, 2000). Molecular-based detection methods for shrimp viruses include normal PCR using degenerate primers (Chang et al., 1993; Lu et al., 1993; Vickers et al., 1992) and nested PCR (Belcher and Young, 1998), among others. Using PCR primers previously reported, we developed a duplex PCR protocol for the simultaneous detection of WSSV and MBV. It provides a convenient tool for screening the said viruses at reduced time and

handling. This protocol was more sensitive than either the non-nested PCR earlier reported for MBV (Belcher and Young, 1998) or the two-step PCR detection of WSSV (Tapay et al., 1999), probably due to some modifications like concentration of PCR reagents and additional PCR cycles used (35 instead of 30 cycles). Moreover, it is also possible that the increased sensitivity of our method was due to the type of DNA used as standard. Direct DNA extraction from infected PL would yield not only the viral DNA but also shrimp DNA, thus making it difficult to get an estimate on the concentration of viral DNA. Thus, plasmids containing cloned target sequences were used as PCR standard in order to have an initial estimate on the concentration of the target DNA. Because of the high sensitivity of the duplex PCR protocol, it may also be useful in confirming early stages of WSSV and MBV infection, when the virus concentration is relatively low or before the manifestation of infection. For MBV detection, the simplest method is based on the microscopic demonstration of the characteristic occlusion bodies produced by the virus. However, this method has its limitations since MBV occlusion bodies can be easily detected from PL13 onwards but are difficult to detect in the early postlarval stages (PL1–12). Also, presence of MBV does not always result in disease and mortalities as this virus is well tolerated by P. monodon in light to moderate infections (Lightner, 1988). Though WSSV is highly pathogenic, its presence can often be detected in apparently healthy postlarvae by PCR (Lo et al., 1996; Magbanua et al., 2000; Otta et al., 2003). Positive WSSV tests at the egg and early PL

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stages may be infrequent due to the inability of the virus to replicate at these stages (Lo et al., 1997 and Tsai et al. (1999). However, positive tests are more frequent at later PL (Otta et al., 2003). In conclusion, the developed duplex PCR protocol offers a more rapid, efficient and stringent screening of postlarval samples since both WSSV and MBV can be detected in a single reaction even at the early stages of infection. Using the developed method, analysis of both MBV and WSSV starting from sample preparation can be completed within 5–6 h. Screening of postlarvae intended for stocking would significantly lessen the risk of disease outbreaks especially in the case of the Philippines wherein stocks are moved between islands regularly.

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