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Prevalence of shrimp viruses in wild Penaeus monodon from Brunei Darussalam
Aquaculture 308 (2010) 71–74
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Prevalence of shrimp viruses in wild Penaeus monodon from Brunei Darussalam Kerry Claydon ⁎, Rahimah Awg Haji Tahir, Hajijah Mohd Said, Mahani Haji Lakim, Wanidawati Tamat Aquatic Animal Health Centre, Department of Fisheries, Ministry of Industry and Primary Resources, Brunei Darussalam
a r t i c l e
i n f o
Article history: Received 26 January 2010 Received in revised form 27 July 2010 Accepted 14 August 2010 Keywords: Penaeus monodon Prawn Shrimp Virus PCR Brunei
a b s t r a c t Over a period of 30 months, wild black tiger shrimp (Penaeus monodon) were collected from the South China Sea in South-east Asia, in an effort to establish specific pathogen free (SPF) stocks of P. monodon in Brunei Darussalam. The viral status of the broodstock was assessed by real-time and conventional polymerase chain reaction (PCR) using DNA and reverse transcribed RNA extracted from the gill, hepatopancreas and pleopods. Histopathology was performed to confirm PCR results. Over 270 P. monodon were collected, screened and spawned. Of the nine viruses assessed, infectious hypodermal and hematopoietic necrosis virus (IHHNV) was most commonly detected (19.6%), followed by monodon baculovirus (MBV) (7.4%), hepatopancreatic parvovirus (HPV) (3.8%), and Mourilyan virus (MoV) (0.9%). The only multiple viral infections found were a combination of IHHNV and MBV (2.2%). Interestingly, the two most virulent viruses for P. monodon, white spot syndrome virus (WSSV) and yellowhead virus (YHV) were not detected in any shrimp. It is concluded that wild P. monodon from Brunei waters have a low prevalence of viral pathogens, which has permitted the development of SPF stocks as a foundation population for selective breeding. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Shrimp farming in the Asia-Pacific region is one of the most lucrative aquaculture sectors (De Silva et al., 2007). Shrimp aquaculture has faced many challenges, when over intensification, environmental degradation, and a variety of other factors led to the emergence and establishment of many viral diseases in Penaeus monodon, the backbone of production in the past (De Silva et al., 2007). Production of P. monodon seed stock has relied mainly on capture of wild reproductive adults. These animals are known to be infected with multiple viral diseases (Flegel, 2006) which have contributed to massive economic losses in the aquaculture industry. To combat disease problems in P. monodon, many countries have introduced specific pathogen free (SPF), non-indigenous Pacific white shrimp, P. vannamei. The Sultanate of Brunei Darussalam is a small country bordering eastern Malaysia on the island of Borneo in South-east Asia and has a coast line adjoining the southern edge of the South China Sea. Since 2000, Brunei Darussalam has been culturing SPF Pacific blue shrimp (Penaeus stylirostris) which are remnants of the IHHNV-resistant Super Shrimp™ lines imported from Mexico (Tang et al., 2000). This species comprises only a tiny fraction (0.08%) of global shrimp aquaculture production (FAO, 2008), and also competes directly with P. vannamei, which has become a low-value commodity now
⁎ Corresponding author. Integrated Aquaculture International, 3303 West Twelfth Street, Hastings, NE 68902-0609, USA. Tel.: + 1 673 895 8930; fax: + 1 673 265 2628. E-mail address: [email protected] (K. Claydon). 0044-8486/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2010.08.015
dominating international markets. To provide a higher-value species for Brunei shrimp farmers, the Department of Fisheries of Brunei Darussalam engaged in a joint project with Integrated Aquaculture International, to develop SPF P. monodon. An essential requirement for redevelopment of P. monodon aquaculture is a source of wild shrimp free of major pathogens. More than 20 viruses have been reported to infect marine shrimp (Bonami, 2008) with nine responsible for substantial economic losses. These include white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), monodon baculovirus (MBV), hepatopancreatic parvovirus (HPV), yellowhead virus (YHV), gill-associated virus (GAV), Taura syndrome virus (TSV), infectious myonecrosis virus (IMNV), and Mourilyan virus (MoV). This study sought to investigate the disease status of local P. monodon broodstock populations in an effort to obtain specific pathogen free (SPF) stocks for domestication and breeding.
2. Materials and methods 2.1. Source of animals P. monodon broodstock were collected from the southern section of the South China Sea in South-east Asia. Commercial trawlers collected the shrimp 3–45 nautical miles from the shore. The broodstock were housed individually in aerated boxes during transport back to shore, where they were transferred to individual tanks in a quarantine facility. After spawning, broodstock were sacrificed to obtain samples for PCR and histology.
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2.2. Nucleic acid extraction Nucleic acid was extracted from small pieces of pleopod, gill, and hepatopancreatic tissue. Tissues were homogenised in lysis buffer (Sigma-Aldrich, Steinheim, Germany) with 10% dithiothreitol (DTT) and incubated at 70 °C for 10 min. Following centrifugation (13,400 ×g), 220 μl of lysate was extracted using the chemistry provided by the XTR1-1 kit (Sigma-Aldrich, Steinheim, Germany) and the CAS1820 X-Tractor gene automated DNA/RNA extraction system (Corbett Robotics, Brisbane, Australia), according to the manufacturer's protocol. Eluted DNA/RNA was used immediately for PCR analysis.
in Davidson's fixative (Humason, 1967) for 48 h before being transferred to 70% ethanol. Following tissue processing and embedding, 5 μm sections were stained with Mayer's hematoxylin and eosin (H&E) (Mayer, 1903). Stained slides were examined with a Nikon Eclipse 80i microscope and photographed digitally using a Nikon Digital Sight DS-Fi1 camera. 3. Results 3.1. Shrimp samples A total of 270 wild P. monodon broodstock were collected between May 2007 and November 2009, with a size range of 101–368 g.
2.3. PCR for virus detection PCR was performed for the detection of nine viruses. Commercial kits (IQ2000 and IQReal) developed by GeneReach (Taichung, Taiwan) were used to detect WSSV, TSV, YHV/GAV, IMNV, and MoV. For HPV, the PCR primers HPV 2F/2R described by Tang et al. (2008) were used; for MBV, the PCR primers MBV 216F/R described by Surachetpong et al. (2005) were used; and for infectious IHHNV, the PCR primers IHHNV 309F/R described by Tang et al. (2007) were used (Table 1). To minimize PCR false-negatives, an internal control was included in all PCR reactions. This involved multiplexing reactions to amplify shrimp DNA. For conventional PCR, HPV and IHHNV incorporated β-actin PCR primers (Sellars et al., 2007) which amplified a 201 bp segment of DNA. For MBV, 18S rRNA PCR primers (Lo et al., 1996) were included to produce an 848 bp amplicon (Table 1). Conventional PCR was performed using Ready-To-Go PCR beads (GE Healthcare, Singapore) with each reaction containing 0.4 μM each primer, 10 mM of Tris–HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 2.5 U Taq DNA polymerase, and 2 μl extracted DNA (100–200 ng). Thermal cycling conditions used for multiplex PCRs employed 35 cycles of 94 °C for 20 s, 55 °C for 20 s, and 72 °C for 30 s, with a final 30 s extension at 72 °C. Conventional PCR for HPV, MBV, IHHNV, GAV and MoV were carried out in a mastercycler gradient thermocycler (Eppendorf, Singapore). For analysis, 10 μl PCR mixture was used in electrophoresis on 1.5% agarose gels containing ethidium bromide at a concentration of 1 mg/ml and visualised under ultraviolet transillumination. Real time PCR for WSSV, TSV, IMNV, and YHV used the RotorGene 6000 (Corbett Robotics, Brisbane, Australia). Pleopods from selected shrimp that were PCR positive for IHHNV were preserved in 70% ethanol and shipped to the Aquatic Pathology Laboratory at the University of Arizona (Tucson, Arizona, USA) for subsequent IHHNV PCR and DNA sequence analysis. A 1.2 kb DNA fragment from nucleotide 2758–4001 of the 4.1 kb IHHNV genome was amplified using IHHNV F1/R1 primers (Tang and Lightner, 2002).
3.2. PCR for virus detection IHHNV was the most common virus detected (14.1%), followed by MBV (7.4%), HPV (3.8%) and MoV (0.9%). The only multiple viral infections found involved IHHNV and MBV (2.2%). Interestingly, the two most virulent viruses for P. monodon, WSSV and YHV, were not detected in any wild broodstock. IMNV and TSV were also absent, although this is probably due to P. monodon not being a common natural host (Srisuvan et al., 2005; Tang et al., 2005). All of the PCRs utilized primers as internal controls to ensure PCR validity. The in-house conventional PCRs multiplexed primers for shrimp β-actin (201 bp) or the 18S rRNA gene (848 bp) with virus specific primers (Fig. 1). DNA sequence analysis of IHHNV, amplified a 1.2 kb genome fragment encoding the IHHNV capsid protein region (315 amino acids) showed the Brunei IHHNV strain to be 98–99% identical to the South-east Asia genotype, with IHHNV Thailand genotype (Genbank accession no. AAM28236) having the highest amino acid sequence identity (99%) (data not shown). 3.3. Histology Histology was useful to confirm virus infections detected by PCR. MBV and HPV infections were easily distinguished in the tubules of the hepatopancreas. HPV was distinguished by basophilic inclusions within enlarged nuclei of tubule epithelial cells. Similar to other reports (Flegel, 2006; Lightner and Redman, 1985) actively dividing E-cells at the distal ends of hepatopancreatic tubules showed the most inclusions. MBV was characterized by spherical occlusion bodies in hepatopancreatocytes, which contained markedly hypertrophied nuclei with single or, more often, multiple eosinophilic occlusion bodies together with chromatin diminution and margination (Fig. 2A).
2.4. Histology As a secondary diagnostic check, histology was also performed on broodstock animals. The cephalothorax was removed and preserved
Table 1 Primer nucleotide sequences used for multiplexed PCR tests. Primer name
Primer sequences
Amplicon size
HPV 2F/2R
5′-GGAAGCCTGTGTTCCTGACT-3′ 5′-CGTCTCCGGATTGCTCTGAT-3′ 5′-AATCCTAGGCGATCTTACCA-3′ 5′-CGTTCGTTGATGAACATCTC-3′ 5′-TCCAACACTTAGTCAAAACCAA-3′ 5′-TGTCTGCTACGATGATTATCCA-3′ 5′-GTGGACATCCGTAAGGACCTGTACG-3′ 5′-CTCCTGCTTGCTGATCCACATCTGC-3′ 5′-TGCCTTATCAGCTACGTTCGATTGTAG-3′ 5′-TTCAGACGTTTTGCAACCATACTTCCC-3′
595 bp
MBV 216F/R IHHNV 309F/R β-actin 201F/R 18S rRNA 143F/145R
216 bp 309 bp 201 bp 848 bp
Fig. 1. Multiplex PCR result for IHHNV, HPV and MBV detection. Lane M: GeneRuler (1 kb); Lane 1: IHHNV positive result (309 bp) with amplified shrimp B-actin (201 bp); Lane 2: HPV positive result (595 bp) with amplified shrimp β-actin (201 bp); Lane 3: MBV positive result (216 bp) with amplified shrimp 18S rRNA (848 bp).
K. Claydon et al. / Aquaculture 308 (2010) 71–74
Fig. 2. H&E stained photomicrographs of the two most common viral infections found in wild P. monodon from Brunei Darussalam. (A) Eosinphilic MBV intranuclear occlusion bodies (arrows) in hepatopancreatocytes. (B) Cowdry type A inclusion (arrow) of IHHNV in the antennal gland.
Although histology effectively confirmed most MBV- and HPV-positive shrimp, some escaped confirmation which was not unexpected with low-level viral infections being difficult to observe using this method. The IHHNV pathognomonic histology of Cowdry type A intranuclear inclusion bodies was observed in the antennal glands of some PCR-positive shrimp but not all (Fig. 2B). The two P. monodon found to be PCR-positive for MoV displayed packets of hypertrophied cells within lymphoid organ spheroids, but no distinctive histology for this pathogen was observed.
4. Discussion This study confirmed the presence of shrimp viruses in wild P. monodon from Brunei Darussalam, although viral prevalence was low. IHHNV is endemic in Asian P. monodon (Flegel, 1997; Lightner, 1993) and was the most common virus infection in wild Brunei P. monodon. IHHNV is often asymptomatic in P. monodon and usually does not cause production losses (Flegel, 2006) and has little or no impact on growth or fecundity (Chayaburakul et al., 2005; Withyachumnarnkul et al., 2006). However, horizontal transmission to other susceptible shrimp species can occur. To assure the SPF status for all common shrimp viruses, testing for IHHNV is essential. The IHHNV genotype was matched closely (99%) with the Thailand IHHNV sequence. This is not surprising given that the
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Brunei Darussalam and Thailand coastlines share the same major water body, the South China Sea. Selection of PCR primers for IHHNV identification is important, as some P. monodon carry non-infectious (genomic) IHHNV (Tang et al., 2007). Histology was a used to complement PCR diagnosis, but characteristic Cowdry type A inclusions were not always observed in IHHNV PCR positive P. monodon tissues. Similar findings have been reported previously (Flegel, 2006; Tang et al., 2003) and other, more sensitive diagnostic methods such as in situ hybridization (ISH) are useful for definitive diagnosis (Tang et al., 2000, 2003; Yang et al., 2007). MBV was the second most common virus detected in 7.4% of wild P. monodon broodstock. This prevalence is considered low in view of previous research in other Asian countries that found MBV prevalence in wild P. monodon broodstock to be 14% in the Philippines (de la Peña et al., 2008), 21% in India (Ramasamy et al., 2000), and 25% in Thailand (Fegan et al., 1991). Similarly, HPV prevalence was also low (3.8%) compared to other Asian countries. HPV is considered to have a worldwide distribution following its discovery in Asia over two decades ago (Chong and Loh, 1984). The choice of diagnostic method may influence virus detection, as this study found histology did not confirm all the PCR-positive results. IMNV is the most recent of the known shrimp viruses to arrive in Asia (NACA/FAO, 2006), and is thought to have been introduced with contaminated P. vannamei stocks from Brazil (Senapin et al., 2007). While P. vannamei is the primary host for IMNV, other species such as P. monodon are susceptible to experimental infection (Tang et al., 2005) although no gross signs or mortalities have been reported. TSV, another major pathogen of P. vannamei, is rarely found naturally in P. monodon, although it has been detected in P. monodon populations from Taiwan (Chang et al., 2004), Thailand, and Eritrea (Srisuvan et al., 2005). Experimental bioassays have shown that low-grade mortalities can occur and that P. monodon can carry asymptomatic infections (Chang et al., 2004; Srisuvan et al., 2005). Neither TSV nor IMNV was detected in the wild Brunei P. monodon screened using realtime PCR and histology. Although P. monodon are not considered to be common hosts of these viruses, their detection and subsequent elimination from breeding stocks is essential. In time, these viruses could become important pathogens for P. monodon due to the tendency of such viruses to establish persistent chronic (carrier) infections (Srisuvan et al., 2005), coupled with the low-fidelity nature of RNA-dependent RNA polymerase (Teng et al., 2006), new strains of these viruses could emerge. Sensitive and specific detection of causative disease agents is a prerequisite for effective disease prevention and management (Teng et al., 2006). The current lack of continuous crustacean cell lines suitable for the isolation and growth of viruses is a major constraint for shrimp disease diagnosis (Claydon and Owens, 2008). For the last three decades, crustacean viral infections have been investigated using in vivo experiments involving infectivity bioassays and histopathology. More recently, DNA-based tools such as PCR have become available for rapid and early detection (Bonami, 2008). PCR has many advantages as a diagnostic tool, with its main strengths being its sensitivity, specificity, and rapid analysis. Some of the disadvantages of PCR, such as generating false-positive/negative results (Claydon et al., 2004; Hsu et al., 1999), missing strain/isolate variations (Tang et al., 2008), and cost, can be counteracted by using other diagnostic methods such as histology. In addition, histology has the advantage of assessing the overall health of the animal, allowing simultaneous detection of multiple pathogens, including new pathogens that cannot be detected by PCR due to lack of genomic data. However the major disadvantage of using histology compared with PCR as a diagnostic tool, is its lessened sensitivity, which allows light infections or carriers to go undetected (Flegel et al., 2004; Lightner and Redman, 1998). Used in combination, these two diagnostic methods complement each other in scope, cost, and sensitivity and provide a more confident and balanced health profile.
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Although PCR and histological screening of wild broodstock provides valuable information for the selection of healthy animals, this screening in itself is not sufficient to produce SPF animals. Two years of continual health screening through secondary and tertiary quarantine processes are required to ensure the SPF candidate offspring remain free of viral agents (Hennig et al., 2005; Pantoja et al., 2005). SPF P. monodon established in Brunei Darussalam have been transferred to a breeding program where strict biosecurity procedures and continual health surveillance are implemented to ensure they remain free of pathogens. Our data show that wild P. monodon from Brunei waters have a low prevalence of viral pathogens, as detected by a combination of PCR and histology. This has permitted the development of SPF stocks as a foundation population for selective breeding. Acknowledgements The authors would like to thank the Director of Fisheries Hajah Hasnah Ibraham for her support and initiation of this project. We are also grateful for the help and assistance provided by the Brunei Darussalam Department of Fisheries officers and staff and Integrated Aquaculture International colleagues. Thank you also for the support provided by Dr Leigh Owens of James Cook University, and Dr Donald Lightner and Dr Kathy Tang of the University of Arizona. This work was financially supported by the Department of Fisheries of the Ministry of Industry and Primary Resources of Brunei Darussalam. References Bonami, J.-R., 2008. Shrimp Viruses. In: Mahy, B.W.J., Regenmortel, M.H.V.v. (Eds.), Encyclopedia of Virology. Academic Press, Oxford, pp. 567–576. Chang, Y.-S., Peng, S.-E., Yu, H.-T., Liu, F.-C., Wang, C.-H., Lo, C.-H., Kou, G.-H., 2004. Genetic and phenotypic variations of isolates of shrimp Taura syndrome virus found in Penaeus monodon and Metapenaeus ensis in Taiwan. J. Gen. Virol. 85, 2963–2968. Chayaburakul, K., Lightner, D.V., Sriurairattana, S., Nelson, K.T., Withyachumnarnkul, B., 2005. Different responses to infectious hypodermal and hematopoietic necrosis virus (IHHNV) in Penaeus monodon and P. vannamei. Dis. Aquat. Organ. 67, 191–200. Chong, Y.C., Loh, H., 1984. Hepatopancreas chlamydial and parvoviral infections of farmed marine prawns in Singapore. Singapore Vet. J. 9, 51–56. Claydon, K., Owens, L., 2008. Attempts at immortalization of crustacean primary cell cultures using human cancer genes. In Vitro Cell. Dev. Biol. 44, 451–457. Claydon, K., Cullen, B., Owens, L., 2004. OIE white spot syndrome virus PCR gives falsepositive results in Cherax quadricarinatus. Dis. Aquat. Organ. 62, 265–268. de la Peña, L.D., Lavilla-Pitogo, C.R., Villar, C.B.R., Paner, M.G., Capulos, G.C., 2008. Prevalence of monodon baculovirus (MBV) in wild shrimp Penaeus monodon in the Philippines. Aquaculture 285, 19–22. De Silva, S.S., Mohan, C.V., Phillips, M.J., 2007. A different form of dumping: The need for a precautionary approach for yet another new species for shrimp farming in Asia, Aquaculture Asia. Network of Aquaculture Centres in Asia-Pacific, Bangkok, pp. 3–5. FAO, 2008. Global aquaculture production (1950–2008) Available online at: http:// www.fao.org/fishery/statistics/global-aquaculture-production/query/en. Cited 11 April 2010. Fegan, D.F., Flegel, T.W., Sriurairatana, S., Waiyakruttha, M., 1991. The occurrence, development and histopathology of monodon baculovirus in Penaeus monodon in southern Thailand. Aquaculture 96, 205–217. Flegel, T.W., 1997. Special topic review: major viral diseases of the black tiger prawn (Penaeus monodon) in Thailand. World J. Microbiol. Biotechnol. 13, 433–442. Flegel, T.W., 2006. Detection of major penaeid shrimp viruses in Asia, a historical perspective with emphasis on Thailand. Aquaculture 258, 1–33. Flegel, T.W., Nielsen, L., Thamavit, V., Kongtim, S., Pasharawipasc, T., 2004. Presence of multiple viruses in non-diseased, cultivated shrimp at harvest. Aquaculture 240, 55–68.
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Contents lists available at ScienceDirect
Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Prevalence of shrimp viruses in wild Penaeus monodon from Brunei Darussalam Kerry Claydon ⁎, Rahimah Awg Haji Tahir, Hajijah Mohd Said, Mahani Haji Lakim, Wanidawati Tamat Aquatic Animal Health Centre, Department of Fisheries, Ministry of Industry and Primary Resources, Brunei Darussalam
a r t i c l e
i n f o
Article history: Received 26 January 2010 Received in revised form 27 July 2010 Accepted 14 August 2010 Keywords: Penaeus monodon Prawn Shrimp Virus PCR Brunei
a b s t r a c t Over a period of 30 months, wild black tiger shrimp (Penaeus monodon) were collected from the South China Sea in South-east Asia, in an effort to establish specific pathogen free (SPF) stocks of P. monodon in Brunei Darussalam. The viral status of the broodstock was assessed by real-time and conventional polymerase chain reaction (PCR) using DNA and reverse transcribed RNA extracted from the gill, hepatopancreas and pleopods. Histopathology was performed to confirm PCR results. Over 270 P. monodon were collected, screened and spawned. Of the nine viruses assessed, infectious hypodermal and hematopoietic necrosis virus (IHHNV) was most commonly detected (19.6%), followed by monodon baculovirus (MBV) (7.4%), hepatopancreatic parvovirus (HPV) (3.8%), and Mourilyan virus (MoV) (0.9%). The only multiple viral infections found were a combination of IHHNV and MBV (2.2%). Interestingly, the two most virulent viruses for P. monodon, white spot syndrome virus (WSSV) and yellowhead virus (YHV) were not detected in any shrimp. It is concluded that wild P. monodon from Brunei waters have a low prevalence of viral pathogens, which has permitted the development of SPF stocks as a foundation population for selective breeding. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Shrimp farming in the Asia-Pacific region is one of the most lucrative aquaculture sectors (De Silva et al., 2007). Shrimp aquaculture has faced many challenges, when over intensification, environmental degradation, and a variety of other factors led to the emergence and establishment of many viral diseases in Penaeus monodon, the backbone of production in the past (De Silva et al., 2007). Production of P. monodon seed stock has relied mainly on capture of wild reproductive adults. These animals are known to be infected with multiple viral diseases (Flegel, 2006) which have contributed to massive economic losses in the aquaculture industry. To combat disease problems in P. monodon, many countries have introduced specific pathogen free (SPF), non-indigenous Pacific white shrimp, P. vannamei. The Sultanate of Brunei Darussalam is a small country bordering eastern Malaysia on the island of Borneo in South-east Asia and has a coast line adjoining the southern edge of the South China Sea. Since 2000, Brunei Darussalam has been culturing SPF Pacific blue shrimp (Penaeus stylirostris) which are remnants of the IHHNV-resistant Super Shrimp™ lines imported from Mexico (Tang et al., 2000). This species comprises only a tiny fraction (0.08%) of global shrimp aquaculture production (FAO, 2008), and also competes directly with P. vannamei, which has become a low-value commodity now
⁎ Corresponding author. Integrated Aquaculture International, 3303 West Twelfth Street, Hastings, NE 68902-0609, USA. Tel.: + 1 673 895 8930; fax: + 1 673 265 2628. E-mail address: [email protected] (K. Claydon). 0044-8486/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2010.08.015
dominating international markets. To provide a higher-value species for Brunei shrimp farmers, the Department of Fisheries of Brunei Darussalam engaged in a joint project with Integrated Aquaculture International, to develop SPF P. monodon. An essential requirement for redevelopment of P. monodon aquaculture is a source of wild shrimp free of major pathogens. More than 20 viruses have been reported to infect marine shrimp (Bonami, 2008) with nine responsible for substantial economic losses. These include white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), monodon baculovirus (MBV), hepatopancreatic parvovirus (HPV), yellowhead virus (YHV), gill-associated virus (GAV), Taura syndrome virus (TSV), infectious myonecrosis virus (IMNV), and Mourilyan virus (MoV). This study sought to investigate the disease status of local P. monodon broodstock populations in an effort to obtain specific pathogen free (SPF) stocks for domestication and breeding.
2. Materials and methods 2.1. Source of animals P. monodon broodstock were collected from the southern section of the South China Sea in South-east Asia. Commercial trawlers collected the shrimp 3–45 nautical miles from the shore. The broodstock were housed individually in aerated boxes during transport back to shore, where they were transferred to individual tanks in a quarantine facility. After spawning, broodstock were sacrificed to obtain samples for PCR and histology.
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2.2. Nucleic acid extraction Nucleic acid was extracted from small pieces of pleopod, gill, and hepatopancreatic tissue. Tissues were homogenised in lysis buffer (Sigma-Aldrich, Steinheim, Germany) with 10% dithiothreitol (DTT) and incubated at 70 °C for 10 min. Following centrifugation (13,400 ×g), 220 μl of lysate was extracted using the chemistry provided by the XTR1-1 kit (Sigma-Aldrich, Steinheim, Germany) and the CAS1820 X-Tractor gene automated DNA/RNA extraction system (Corbett Robotics, Brisbane, Australia), according to the manufacturer's protocol. Eluted DNA/RNA was used immediately for PCR analysis.
in Davidson's fixative (Humason, 1967) for 48 h before being transferred to 70% ethanol. Following tissue processing and embedding, 5 μm sections were stained with Mayer's hematoxylin and eosin (H&E) (Mayer, 1903). Stained slides were examined with a Nikon Eclipse 80i microscope and photographed digitally using a Nikon Digital Sight DS-Fi1 camera. 3. Results 3.1. Shrimp samples A total of 270 wild P. monodon broodstock were collected between May 2007 and November 2009, with a size range of 101–368 g.
2.3. PCR for virus detection PCR was performed for the detection of nine viruses. Commercial kits (IQ2000 and IQReal) developed by GeneReach (Taichung, Taiwan) were used to detect WSSV, TSV, YHV/GAV, IMNV, and MoV. For HPV, the PCR primers HPV 2F/2R described by Tang et al. (2008) were used; for MBV, the PCR primers MBV 216F/R described by Surachetpong et al. (2005) were used; and for infectious IHHNV, the PCR primers IHHNV 309F/R described by Tang et al. (2007) were used (Table 1). To minimize PCR false-negatives, an internal control was included in all PCR reactions. This involved multiplexing reactions to amplify shrimp DNA. For conventional PCR, HPV and IHHNV incorporated β-actin PCR primers (Sellars et al., 2007) which amplified a 201 bp segment of DNA. For MBV, 18S rRNA PCR primers (Lo et al., 1996) were included to produce an 848 bp amplicon (Table 1). Conventional PCR was performed using Ready-To-Go PCR beads (GE Healthcare, Singapore) with each reaction containing 0.4 μM each primer, 10 mM of Tris–HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 2.5 U Taq DNA polymerase, and 2 μl extracted DNA (100–200 ng). Thermal cycling conditions used for multiplex PCRs employed 35 cycles of 94 °C for 20 s, 55 °C for 20 s, and 72 °C for 30 s, with a final 30 s extension at 72 °C. Conventional PCR for HPV, MBV, IHHNV, GAV and MoV were carried out in a mastercycler gradient thermocycler (Eppendorf, Singapore). For analysis, 10 μl PCR mixture was used in electrophoresis on 1.5% agarose gels containing ethidium bromide at a concentration of 1 mg/ml and visualised under ultraviolet transillumination. Real time PCR for WSSV, TSV, IMNV, and YHV used the RotorGene 6000 (Corbett Robotics, Brisbane, Australia). Pleopods from selected shrimp that were PCR positive for IHHNV were preserved in 70% ethanol and shipped to the Aquatic Pathology Laboratory at the University of Arizona (Tucson, Arizona, USA) for subsequent IHHNV PCR and DNA sequence analysis. A 1.2 kb DNA fragment from nucleotide 2758–4001 of the 4.1 kb IHHNV genome was amplified using IHHNV F1/R1 primers (Tang and Lightner, 2002).
3.2. PCR for virus detection IHHNV was the most common virus detected (14.1%), followed by MBV (7.4%), HPV (3.8%) and MoV (0.9%). The only multiple viral infections found involved IHHNV and MBV (2.2%). Interestingly, the two most virulent viruses for P. monodon, WSSV and YHV, were not detected in any wild broodstock. IMNV and TSV were also absent, although this is probably due to P. monodon not being a common natural host (Srisuvan et al., 2005; Tang et al., 2005). All of the PCRs utilized primers as internal controls to ensure PCR validity. The in-house conventional PCRs multiplexed primers for shrimp β-actin (201 bp) or the 18S rRNA gene (848 bp) with virus specific primers (Fig. 1). DNA sequence analysis of IHHNV, amplified a 1.2 kb genome fragment encoding the IHHNV capsid protein region (315 amino acids) showed the Brunei IHHNV strain to be 98–99% identical to the South-east Asia genotype, with IHHNV Thailand genotype (Genbank accession no. AAM28236) having the highest amino acid sequence identity (99%) (data not shown). 3.3. Histology Histology was useful to confirm virus infections detected by PCR. MBV and HPV infections were easily distinguished in the tubules of the hepatopancreas. HPV was distinguished by basophilic inclusions within enlarged nuclei of tubule epithelial cells. Similar to other reports (Flegel, 2006; Lightner and Redman, 1985) actively dividing E-cells at the distal ends of hepatopancreatic tubules showed the most inclusions. MBV was characterized by spherical occlusion bodies in hepatopancreatocytes, which contained markedly hypertrophied nuclei with single or, more often, multiple eosinophilic occlusion bodies together with chromatin diminution and margination (Fig. 2A).
2.4. Histology As a secondary diagnostic check, histology was also performed on broodstock animals. The cephalothorax was removed and preserved
Table 1 Primer nucleotide sequences used for multiplexed PCR tests. Primer name
Primer sequences
Amplicon size
HPV 2F/2R
5′-GGAAGCCTGTGTTCCTGACT-3′ 5′-CGTCTCCGGATTGCTCTGAT-3′ 5′-AATCCTAGGCGATCTTACCA-3′ 5′-CGTTCGTTGATGAACATCTC-3′ 5′-TCCAACACTTAGTCAAAACCAA-3′ 5′-TGTCTGCTACGATGATTATCCA-3′ 5′-GTGGACATCCGTAAGGACCTGTACG-3′ 5′-CTCCTGCTTGCTGATCCACATCTGC-3′ 5′-TGCCTTATCAGCTACGTTCGATTGTAG-3′ 5′-TTCAGACGTTTTGCAACCATACTTCCC-3′
595 bp
MBV 216F/R IHHNV 309F/R β-actin 201F/R 18S rRNA 143F/145R
216 bp 309 bp 201 bp 848 bp
Fig. 1. Multiplex PCR result for IHHNV, HPV and MBV detection. Lane M: GeneRuler (1 kb); Lane 1: IHHNV positive result (309 bp) with amplified shrimp B-actin (201 bp); Lane 2: HPV positive result (595 bp) with amplified shrimp β-actin (201 bp); Lane 3: MBV positive result (216 bp) with amplified shrimp 18S rRNA (848 bp).
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Fig. 2. H&E stained photomicrographs of the two most common viral infections found in wild P. monodon from Brunei Darussalam. (A) Eosinphilic MBV intranuclear occlusion bodies (arrows) in hepatopancreatocytes. (B) Cowdry type A inclusion (arrow) of IHHNV in the antennal gland.
Although histology effectively confirmed most MBV- and HPV-positive shrimp, some escaped confirmation which was not unexpected with low-level viral infections being difficult to observe using this method. The IHHNV pathognomonic histology of Cowdry type A intranuclear inclusion bodies was observed in the antennal glands of some PCR-positive shrimp but not all (Fig. 2B). The two P. monodon found to be PCR-positive for MoV displayed packets of hypertrophied cells within lymphoid organ spheroids, but no distinctive histology for this pathogen was observed.
4. Discussion This study confirmed the presence of shrimp viruses in wild P. monodon from Brunei Darussalam, although viral prevalence was low. IHHNV is endemic in Asian P. monodon (Flegel, 1997; Lightner, 1993) and was the most common virus infection in wild Brunei P. monodon. IHHNV is often asymptomatic in P. monodon and usually does not cause production losses (Flegel, 2006) and has little or no impact on growth or fecundity (Chayaburakul et al., 2005; Withyachumnarnkul et al., 2006). However, horizontal transmission to other susceptible shrimp species can occur. To assure the SPF status for all common shrimp viruses, testing for IHHNV is essential. The IHHNV genotype was matched closely (99%) with the Thailand IHHNV sequence. This is not surprising given that the
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Brunei Darussalam and Thailand coastlines share the same major water body, the South China Sea. Selection of PCR primers for IHHNV identification is important, as some P. monodon carry non-infectious (genomic) IHHNV (Tang et al., 2007). Histology was a used to complement PCR diagnosis, but characteristic Cowdry type A inclusions were not always observed in IHHNV PCR positive P. monodon tissues. Similar findings have been reported previously (Flegel, 2006; Tang et al., 2003) and other, more sensitive diagnostic methods such as in situ hybridization (ISH) are useful for definitive diagnosis (Tang et al., 2000, 2003; Yang et al., 2007). MBV was the second most common virus detected in 7.4% of wild P. monodon broodstock. This prevalence is considered low in view of previous research in other Asian countries that found MBV prevalence in wild P. monodon broodstock to be 14% in the Philippines (de la Peña et al., 2008), 21% in India (Ramasamy et al., 2000), and 25% in Thailand (Fegan et al., 1991). Similarly, HPV prevalence was also low (3.8%) compared to other Asian countries. HPV is considered to have a worldwide distribution following its discovery in Asia over two decades ago (Chong and Loh, 1984). The choice of diagnostic method may influence virus detection, as this study found histology did not confirm all the PCR-positive results. IMNV is the most recent of the known shrimp viruses to arrive in Asia (NACA/FAO, 2006), and is thought to have been introduced with contaminated P. vannamei stocks from Brazil (Senapin et al., 2007). While P. vannamei is the primary host for IMNV, other species such as P. monodon are susceptible to experimental infection (Tang et al., 2005) although no gross signs or mortalities have been reported. TSV, another major pathogen of P. vannamei, is rarely found naturally in P. monodon, although it has been detected in P. monodon populations from Taiwan (Chang et al., 2004), Thailand, and Eritrea (Srisuvan et al., 2005). Experimental bioassays have shown that low-grade mortalities can occur and that P. monodon can carry asymptomatic infections (Chang et al., 2004; Srisuvan et al., 2005). Neither TSV nor IMNV was detected in the wild Brunei P. monodon screened using realtime PCR and histology. Although P. monodon are not considered to be common hosts of these viruses, their detection and subsequent elimination from breeding stocks is essential. In time, these viruses could become important pathogens for P. monodon due to the tendency of such viruses to establish persistent chronic (carrier) infections (Srisuvan et al., 2005), coupled with the low-fidelity nature of RNA-dependent RNA polymerase (Teng et al., 2006), new strains of these viruses could emerge. Sensitive and specific detection of causative disease agents is a prerequisite for effective disease prevention and management (Teng et al., 2006). The current lack of continuous crustacean cell lines suitable for the isolation and growth of viruses is a major constraint for shrimp disease diagnosis (Claydon and Owens, 2008). For the last three decades, crustacean viral infections have been investigated using in vivo experiments involving infectivity bioassays and histopathology. More recently, DNA-based tools such as PCR have become available for rapid and early detection (Bonami, 2008). PCR has many advantages as a diagnostic tool, with its main strengths being its sensitivity, specificity, and rapid analysis. Some of the disadvantages of PCR, such as generating false-positive/negative results (Claydon et al., 2004; Hsu et al., 1999), missing strain/isolate variations (Tang et al., 2008), and cost, can be counteracted by using other diagnostic methods such as histology. In addition, histology has the advantage of assessing the overall health of the animal, allowing simultaneous detection of multiple pathogens, including new pathogens that cannot be detected by PCR due to lack of genomic data. However the major disadvantage of using histology compared with PCR as a diagnostic tool, is its lessened sensitivity, which allows light infections or carriers to go undetected (Flegel et al., 2004; Lightner and Redman, 1998). Used in combination, these two diagnostic methods complement each other in scope, cost, and sensitivity and provide a more confident and balanced health profile.
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Although PCR and histological screening of wild broodstock provides valuable information for the selection of healthy animals, this screening in itself is not sufficient to produce SPF animals. Two years of continual health screening through secondary and tertiary quarantine processes are required to ensure the SPF candidate offspring remain free of viral agents (Hennig et al., 2005; Pantoja et al., 2005). SPF P. monodon established in Brunei Darussalam have been transferred to a breeding program where strict biosecurity procedures and continual health surveillance are implemented to ensure they remain free of pathogens. Our data show that wild P. monodon from Brunei waters have a low prevalence of viral pathogens, as detected by a combination of PCR and histology. This has permitted the development of SPF stocks as a foundation population for selective breeding. Acknowledgements The authors would like to thank the Director of Fisheries Hajah Hasnah Ibraham for her support and initiation of this project. We are also grateful for the help and assistance provided by the Brunei Darussalam Department of Fisheries officers and staff and Integrated Aquaculture International colleagues. Thank you also for the support provided by Dr Leigh Owens of James Cook University, and Dr Donald Lightner and Dr Kathy Tang of the University of Arizona. This work was financially supported by the Department of Fisheries of the Ministry of Industry and Primary Resources of Brunei Darussalam. References Bonami, J.-R., 2008. Shrimp Viruses. In: Mahy, B.W.J., Regenmortel, M.H.V.v. (Eds.), Encyclopedia of Virology. Academic Press, Oxford, pp. 567–576. Chang, Y.-S., Peng, S.-E., Yu, H.-T., Liu, F.-C., Wang, C.-H., Lo, C.-H., Kou, G.-H., 2004. Genetic and phenotypic variations of isolates of shrimp Taura syndrome virus found in Penaeus monodon and Metapenaeus ensis in Taiwan. J. Gen. Virol. 85, 2963–2968. Chayaburakul, K., Lightner, D.V., Sriurairattana, S., Nelson, K.T., Withyachumnarnkul, B., 2005. Different responses to infectious hypodermal and hematopoietic necrosis virus (IHHNV) in Penaeus monodon and P. vannamei. Dis. Aquat. Organ. 67, 191–200. Chong, Y.C., Loh, H., 1984. Hepatopancreas chlamydial and parvoviral infections of farmed marine prawns in Singapore. Singapore Vet. J. 9, 51–56. Claydon, K., Owens, L., 2008. Attempts at immortalization of crustacean primary cell cultures using human cancer genes. In Vitro Cell. Dev. Biol. 44, 451–457. Claydon, K., Cullen, B., Owens, L., 2004. OIE white spot syndrome virus PCR gives falsepositive results in Cherax quadricarinatus. Dis. Aquat. Organ. 62, 265–268. de la Peña, L.D., Lavilla-Pitogo, C.R., Villar, C.B.R., Paner, M.G., Capulos, G.C., 2008. Prevalence of monodon baculovirus (MBV) in wild shrimp Penaeus monodon in the Philippines. Aquaculture 285, 19–22. De Silva, S.S., Mohan, C.V., Phillips, M.J., 2007. A different form of dumping: The need for a precautionary approach for yet another new species for shrimp farming in Asia, Aquaculture Asia. Network of Aquaculture Centres in Asia-Pacific, Bangkok, pp. 3–5. FAO, 2008. Global aquaculture production (1950–2008) Available online at: http:// www.fao.org/fishery/statistics/global-aquaculture-production/query/en. Cited 11 April 2010. Fegan, D.F., Flegel, T.W., Sriurairatana, S., Waiyakruttha, M., 1991. The occurrence, development and histopathology of monodon baculovirus in Penaeus monodon in southern Thailand. Aquaculture 96, 205–217. Flegel, T.W., 1997. Special topic review: major viral diseases of the black tiger prawn (Penaeus monodon) in Thailand. World J. Microbiol. Biotechnol. 13, 433–442. Flegel, T.W., 2006. Detection of major penaeid shrimp viruses in Asia, a historical perspective with emphasis on Thailand. Aquaculture 258, 1–33. Flegel, T.W., Nielsen, L., Thamavit, V., Kongtim, S., Pasharawipasc, T., 2004. Presence of multiple viruses in non-diseased, cultivated shrimp at harvest. Aquaculture 240, 55–68.
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