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An evidence on trans-ovarian transmission of Monodon baculovirus (MBV) infection in Penaeus monodon
Aquaculture 443 (2015) 5–10
Contents lists available at ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aqua-online
An evidence on trans-ovarian transmission of Monodon baculovirus (MBV) infection in Penaeus monodon Duangkhaetita Kanjanasopa a, Pattira Pongtippatee a, Rapeepun Vanichviriyakit b, Somjai Wongtripop c, Padmaja Jayaprasad Pradeep b, Boonsirm Withyachumnarnkul b,c,⁎ a b c
Aquatic Animal Biotechnology Research Center, Faculty of Science and Industrial Technology, Prince of Songkla University, Surat Thani 84000, Thailand Department of Anatomy and Centex Shrimp, Chalerm Prakiat Building 4th Floor, Faculty of Science, Mahidol University, Rama 6 Rd, Bangkok 10400, Thailand Shrimp Genetic Improvement Center, Surat Thani 84110, Thailand
a r t i c l e
i n f o
Article history: Received 7 January 2015 Received in revised form 22 February 2015 Accepted 23 February 2015 Available online 7 March 2015 Keywords: Monodon baculovirus Trans-ovarian transmission Penaeus monodon Polyhedrin
a b s t r a c t Monodon baculovirus (MBV) causes slow growth of the shrimp being due to its infection in the hepatopancreas, the organ producing digestive enzymes and for nutrient storage. It has long been documented that the virus is transmitted from broodstock to offspring via contamination in rearing water by feces of the infected broodstock that contained sloughing-off damaged hepatopancreatic cells containing MBV. As a management practice, the washing of eggs and nauplii with disinfectant has been recommended to eliminate MBV that are attached to their surface and thus prevent the infection at the later development stage of the shrimp. However, we detected the MBV infection in postlarvae and juveniles of the black tiger shrimp Penaeus monodon, even after our repeated attempts to eliminate the virus by washing the eggs and nauplii with povidone iodine as disinfectant. Therefore we used MBV-infected broodstock to identify the cause of this problem by tracing the presence of MBV in hepatopancreas and ovary of the broodstock, eggs, nauplii and postlarvae, using histology with hematoxylin–eosin staining, polymerase chain reaction (PCR), in situ hybridization (ISH) and immunohistochemistry (IHC) specific for polyhedrin, a protein produced by MBV. We found all the ovaries collected from the broodstock, which were detected MBV-positive in the hepatopancreas by histology, PCR and ISH, were also PCR-positive. By ISH, positive signals were detected in the cell membrane, cytoplasm and nuclear membrane of the oocytes. The eggs and nauplii from the MBV-positive broodstock were also positive by PCR, in both “wash” and “no-wash” specimens. By ISH, positive signals were detected in ooplasm and subcuticular region of nauplii, as well as inside its body. Using IHC, positive signals were detected inside the body and appendage of the nauplius. Taken all these together, it is most likely that MBV could be vertically transmitted through trans-ovarian route. Hence, simply washing the eggs and nauplii with disinfectant may not be an adequate procedure to eliminate the MBV infection in offspring from MBV-infected broodstock. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Monodon baculovirus (MBV), an occluded double-stranded DNA virus of the family Baculoviridiae, could infect several species of penaeid shrimp, including the black tiger shrimp Penaeus monodon, from early to adult stages (Lightner and Redman, 1981; Lin, 1989; Liao et al., 1992; Rajendran et al., 2012). Infection with this virus usually does not cause shrimp mortality but high level of infection could result in slow growth of the shrimp and thus low production at harvest (Fegan et al., 1991; Flegel et al., 2004). The virus produces a matrix composed of polyhedrin protein surrounding its virion and thus is an occluded type. However, detailed study of MBV nucleocapsid and envelope structure has led a group of investigators to suggest that the virus should be grouped as a ⁎ Corresponding author at: Centex Shrimp, Faculty of Science, Mahidol University, 272 Rama 6 Rd, Bangkok 10400, Thailand. Tel.: +66 2 201 5871; fax: +66 2 354 7344. E-mail address: [email protected] (B. Withyachumnarnkul).
http://dx.doi.org/10.1016/j.aquaculture.2015.02.041 0044-8486/© 2015 Elsevier B.V. All rights reserved.
non-occluded one and renamed as singly envelope nuclear polyhydrosis virus from P. monodon or PnSNPV (Mari et al., 1993). The main target organ of infection by MBV in shrimp is hepatopancreas and its detection is easily observed by routine histology with hematoxylin–eosin (H&E) stain, in which the nuclei of the infected hepatopancreatic cells will reveal small multiple spherical acidophilic inclusions (Lightner and Redman, 1981). Quick diagnosis could be done by squash mounts of shrimp hepatopancreas and stained with malachite green, in which the occlusion bodies could be clearly observed (Lightner, 1985). In standard hatchery procedures, female broodstock are screened out for this virus prior to spawning by microscopic examination of fresh feces stained with malachite green (Lightner, 1996) or by polymerase chain reaction (PCR) specific for MBV. Early report suggests that MBV infection in the shrimp fry was transmitted from broodstock via feces contaminated with sloughing-off hepatopancreatic cells containing MBV occlusive bodies (Johnson and
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D. Kanjanasopa et al. / Aquaculture 443 (2015) 5–10
Lightner, 1988). The virus attach to the external surface of the eggs and nauplii. Since the nauplii have no fully developed mouth, the oral intake of the virus particles is rarely possible, but once they reach the zoeal stage and begin to have oral feed intake, the virus could reach the internal organs and slowly invade the hepatopancreas (Chen et al., 1989). Therefore, proper washing of eggs and nauplii thoroughly with disinfectants could free MBV infection at the later developmental stages of the fry. However, it was also reported that the disinfection procedure either requires high concentrations or a combination of more than one type of disinfectant (Chen et al., 1992; Spann et al., 1993; Manisseri et al., 1999). Although it is possible that the inclusion bodies function as a protector of MBV virions against the disinfectants (Mari et al., 1993), one cannot rule out another possibility that a fraction of MBV might be located intracellularly in the eggs and nauplii and thus escape the destruction by disinfectants. To investigate this possibility, we have traced the presence of gene and protein of polyhedrin, which is produced by MBV, in the ovaries of MBV-infected broodstock, and in eggs, nauplii and postlarvae (PL) generated by the broodstock, and the results reported herein. 2. Materials and methods 2.1. Sample collection Samples were collected from a commercial P. monodon hatchery. Feces of individual broodstocks were checked for the presence of MBV by PCR, and both MBV-negative and MBV-positive broodstocks were allowed to spawn individually in separate tanks. The eggs, nauplii, larvae and PLs were subsequently produced under standard hatchery production. The eggs and nauplii were divided into “wash” and “no-wash” groups. The “wash” group was those that were washed five times with povidone iodine (10 ppm) solution and three times with clean seawater, one-min each; and both disinfection and washing procedures were completely avoided for “no-wash” group. Samples collected from both MBV-negative and MBV-positive broodstocks and their respective fry at various stages were subjected to polyhedrin detections at both gene and protein levels, by in situ hybridization (ISH) and immunohistochemistry (IHC), respectively. Some samples were subjected to routine histology procedure with H&E staining and PCR detection for MBV as well (Table 1). Samples for routine histology, ISH and IHC were fixed in Davidson's fixatives and those for PCR were fixed in absolute ethanol. 2.2. Routine histology Samples were processed through paraffin sectioning with H&E staining (Lightner, 1996). Sections were observed particularly for the presence of multiple acidophilic occlusion bodies, a typical feature of Table 1 Sample collections and techniques performed. Number of determinations = 6. Sample Broodstock Feces Hepatopancreas Ovary Eggs (50 eggs/determinationa) “Wash” “No-wash” Nauplii (50 nauplii/determinationa) “Wash” “No-wash” PLs (20 PLs/determinationa) Derived from “wash” nauplii Derived from “no-wash” nauplii a
For PCR.
For PCR Histology, PCR, and ISH Histology, PCR, and ISH PCR and ISH PCR and ISH PCR, ISH and IHC PCR, ISH and IHC Histology and PCR Histology and PCR
MBV infection, in nuclei of the hepatopancreas of the broodstock and PLs, and ovaries of the broodstock. 2.3. PCR method All samples for PCR detection were kept in absolute ethanol and stored at −20 °C until use. DNA extraction was preceded by discarding absolute ethanol and the samples were grinded in 600 μl of lysis buffer containing 50 mM Tris–HCl, pH 9.0, 50 mM EDTA, 1.2 M NaCl and 0.2% Triton-X100 and heated at 95 °C for 15 min. After chloroform/isoamyl alcohol extraction, DNA was precipitated with absolute ethanol and its concentration and quality were measured by spectophotometric analysis at 260 and 280 nm. The amount used for each PCR determination was 50 ng DNA/μl. Nested PCR for detection of MBV was employed according to the method described by Belcher and Young (1998). Primers were designed based on sequence of the MBV polyhedrin gene (EU251062) (Chaivisuthangkura et al., 2008). Primary PCR and nested PCR amplification was performed with RBC Blue Taq Mastermix (RBC Bioscience). The primary PCR reaction was carried out in a 20 μl reaction mixture consisting of 100 ng DNA template, 10 μl of 2 × RBC Blue Taq Mastermix (final concentration: 10 mM KCl, 1.5 mM MgCl2 , 10 mM Tris–HCl (pH 8.3), 0.1 mg/ml BSA, 10 mM (NH4) 2 SO 4 , 0.2 mM dNTP mix, and 0.15 U/μl RBC Taq DNA polymerase), and 0.5 μM of pMBV-F (5′ATGTTCGACGATAGCATGAT-3′) and pMBV-R (5′-TTATTCATTTG TATGATGCG-3′). The cycle conditions for primary PCR amplification consist of: one cycle of 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 50 °C for 30 s, 72 °C for 90 s, and one cycle of 72 °C for 5 min. Nested PCR was carried out in a 25 μl reaction mixture consisting of 1 μl of the primary PCR reaction as the template for the nested PCR, 12.5 μl of 2 × RBC Blue Taq Mastermix (the final concentration was the same as above), and 0.5 μM of each internal (nested) primers as pMBV-NF(5′-CCACACAACATGCTTTGGAC-3′) and pMBV-NR (5′-GGCTAGACGCACACCAAGAT-3′). The second round amplification consists of: one cycle of 94 °C for 5 min; 25 cycles of 94 °C for 30 s, 50 °C for 30 s, 72 °C for 40 s, and one cycle of 72 °C for 5 min. Amplification products of the primary and nested PCR were analyzed by running at 1% of agarose gel and approximate sizes of the products were 1359 and 399 bp, respectively. 2.4. In situ hybridization All samples fixed with Davidson's fixative were kept at room temperature for 24 h before being transferred to 50% ethanol and stored at 4 °C until use. A labeled DNA probe to target the MBV polyhedrin gene was prepared using a commercial PCR DIG-labeling mix (Roche Molecular Biochemicals) according to the manufacturer's instructions. Nested PCR primers and protocol for the labeling reaction were produced as described above. DNA of MBV infected shrimp was used as template. The labeled probe used in a standard in situ hybridization assay method was adapted from the Genius™ Nonradioactive in situ Hybridization Application Manual by Boehringer Mannheim Biochemicals. The tissues were processed through paraffin sectioning and the paraffin-embedded tissues were placed on the tissue paper before placing them inside an oven (45–50 °C) for overnight. They were deparaffinized in fresh xylene 3 times for 5 min before re-hydration with series of alcohol followed by ethanol treatment at various concentrations in an ascending order; 100%, 95%, 80%, 70%, and 50% for 5 min each. After washing with fresh distilled water for 5 min, the sections were placed in TNE solution containing 50 mM Tris–HCl, 10 mM NaCl, 1 mM EDTA, (pH 7.0) for 5 min. Tissue sections were treated with proteinase K solution and incubated for 30 min at 37 °C in humid chamber. The sections were re-fixed with ice cold 0.4% formaldehyde solution for 5 min and immersed in distilled water for 5 min. Then, they were incubated at 37 °C for at least 10 min with pre-hybridization buffer. The buffer was carefully removed and hybridization buffer containing DIG-
D. Kanjanasopa et al. / Aquaculture 443 (2015) 5–10 Table 2 The presence of Monodon baculovirus in broodstock and nauplii as detected by polymerase chain reaction (PCR) and in situ hybridization (ISH) method. Hep, hepatopancreas; Ov, ovary. Batch no.
1 2 3 4 5 6
Broodstock Hep
Nauplius “Wash”
Ov
PCR
ISH PCR
ISH PCR
+ − + + − +
+ − + + + +
+ − + + − +
+ − − + − +
− − + − − −
PL “No-wash”
ISH PCR + − + + − +
− − + − − −
Derive from “wash” nauplii
Derive from “no-wash” nauplii
ISH
PCR
PCR
+ − + − − +
+ − + + − +
+ − + − − +
labeled probe was added. The sections were covered with cover slips and incubated at 42 °C overnight in humid chamber. The cover slips were removed and the sections were washed in washing solution. The
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detection system was carried out by immunological method with NBT/BCIP system (Roche) according to the manufacturer's instructions. The nuclei were counter stained with 0.5% Bismarck brown Y. The tissues were then observed under light microscope. Negative controls were processed identically without the probe. 2.5. Immunohistochemistry The paraffin sections of the samples were de-paraffinized and rehydrated as described above. The sections were incubated with blocking reagent [4% bovine serum albumin and 10% normal goat serum in phosphate buffer saline containing 0.01% Tween 20 (PBST)] for 1 h and then with anti-polyhedrin antibody (MBV5-3H monoclonal antibody) (Satidkanitkul et al., 2005; Boonsanongchokying et al., 2006) at a dilution of 1:200 for 3 h at room temperature. The sections were washed 3 times with PBST and incubated with horseradish peroxidase conjugated goat anti-mouse IgG (Sigma-Aldrich) (1:500) for 1 h at room temperature. The sections were extensively washed with PBST and the antigen-antibody binding signal was developed using VECTOR
Fig. 1. Histology with H&E staining of samples of hepatopancreas (a) and ovary (b) of MBV-infected Penaeus monodon broodstock showing acidophilic multiple occlusion bodies in the nuclei of the hepatopancreas (a, arrow), but not in the ovary. By polymerase chain reaction, gel electrophoresis shows positive signal in both hepatopancreas (c, lanes 3, 5 and 6) and ovary (d, lanes 3, 6 and 9). M, DNA markers; P, positive control; Lane 1, negative control; Lanes 2–10, samples.
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NovaRED Peroxidase Substrate Kit (Vector laboratories). The negative control was performed in parallel but without the primary antibody. 3. Results and discussion 3.1. MBV transmission from broodstock to PLs Using PCR, the 4/6 of hepatopancreas and 3/6 of ovaries under the study were shown positive for MBV (Table 2). Three broodstock that had MBV-positive in the hepatopancreas also were MBV-positive in the ovaries. Using ISH, one more positive case (that was PCR-negative) was detected in the hepatopancreas, as well as in the ovary. Not all broodstock with MBV-positive ovaries by PCR method produced MBVpositive nauplii. By PCR detection, only 1/3 of the MBV-positive ovaries generated MBV-positive nauplii from “wash” specimens, while using ISH, 4/4 of the MBV-positive ovaries generated MBV-positive nauplii in “wash” specimen. In “no-wash” specimens, only 1/6 nauplius batches were MBV-positive by PCR, and the positive batch was not from the same broodstock that was MBV-positive in the ovary; however, by using ISH, 3/4 of broodstocks with MBV-positive generated MBVpositive nauplii. When nauplii, both “wash” and “no-wash” specimens, grew up to PLs, they were again determined for the presence of MBV by PCR and histology. The results from the PCR detection at this stage were correlated with those of nauplii detected by ISH. The above results suggested that (1) ISH was more sensitive than PCR in detecting MBV; (2) broodstock with MBV-positive in the ovary, by either PCR or ISH, generated MBV-positive nauplii; and (3) washing with povidone iodine in the eggs and nauplii could not completely eliminate MBV infection in the nauplii, as well as in PLs.
3.2. Localization of MBV By routine histology of hepatopancreas of broodstock and PLs, typical acidophilic multiple occlusion bodies in the nuclei were clearly observed; and the histological features were corresponded to the PCR results. In the ovaries, no occlusion bodies were observed in the histology of any specimen, despite 3/6 of the ovaries examined were MBVpositive by PCR. Fig. 1 shows histology with H&E staining of samples of hepatopancreas and ovary of MBV-infected broodstock, as well as a sample of gel electrophoresis for MBV detection by PCR, which shows a clear positive result in some ovarian samples that did not contain occlusion bodies. In the hepatopancreas, using ISH, the positive signals were detected in the nuclei of hepatopancreatic cells (Fig. 2b), while in the ovary, the positive signals were observed in the cell membrane, cytoplasm and nuclear membrane of oocytes (Fig. 2c). The size of the signals in the ovary was much smaller than those in the hepatopancreas. The reason behind this weaker signal detection of polyhedrin gene of MBV could be because of the lesser number of polyhedrin gene copies in the ovary than that in the hepatopancreas. The results also suggest that the absence of occlusion bodies in the ovary does not rule out MBV infection in the tissue. It is possible that in order to produce occlusion bodies, either sufficient number of MBV copies must exist or certain conditions in the host cells are required. Thus far, there has not been any report of MBV occlusion bodies in any other tissues besides hepatopancreas (Rajendran et al., 2012). There were no positive signals from the egg surface of both “wash” and “no-wash” eggs, when analyzed using ISH, however, positive signals were observed inside the ooplasm of the eggs (Fig. 3a), suggesting that MBV was inside the egg. Likewise, in nauplii, both “wash” and “no-
Fig. 2. In situ hybridization of the hepatopancreas and ovary of MBV-infected Penaeus monodon using specific probe for polyhedrin gene. The picture shows the hepatopancreas without probe (a) and with probe (b). Positive signals appear as black dots inside the nuclei of the hepatopancreatic cells. In the ovary (c), positive signals were detected as black dots on the cell membrane, inside the cytoplasm and on the nuclear membrane.
D. Kanjanasopa et al. / Aquaculture 443 (2015) 5–10
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Fig. 3. In situ hybridization of the egg (a) and nauplius (b) of MBV-infected Penaeus monodon using specific probe for polyhedrin gene. Positive signals were detected in the ooplasm and subcuticular epithelium, as well as inside the body of the nauplius.
wash” specimens, positive signals were observed underneath the subcuticular epithelium and inside the nauplius body (Fig. 3b). In addition, by IHC, positive signal was also observed inside the body of the nauplius (Fig. 4). Since the nauplius has not yet developed its mouth and the possible entry of MBV through oral route is less certain, hence the presence of MBV inside its body strongly suggest that MBV was derived from the egg and ovary of the broodstock. It is likely possible that the dose of povidone iodine used in this study might not be strong enough to destroy MBV on the surface of the eggs and nauplii, but the finding of positive expression of MBV polyhedrin inside the eggs and nauplii as revealed from ISH and IHC suggests that washing the eggs with disinfectant is not a reliable prophylactic measurement for eliminating MBV from the infected specimens. This study strongly suggests that MBV could be vertically transmitted via trans-ovarian route. However, the results of this study do not deny the fact that MBV infection could be transmitted horizontally as it has been proven previously that the transmission could occur by feeding MBV-contaminated hepatopancreas to PLs (Chen et al., 1992; Paynter et al., 1992). In practical point of view, this study revealed that disinfecting and washing eggs/nauplii with the purpose of “complete elimination” of MBV from their surface cannot be fully guaranteed. Hence, we strongly recommend revised strategies for the prevention of MBV in penaeid shrimp by eliminating all MBV-infected broodstock from the hatchery. Acknowledgments The study was supported by Prince of Songkla University, grant no. S&T550412S. References Belcher, C.R., Young, P.R., 1998. Colouremetric PCR-based detection of monodon baculovirus in whole Penaeus monodon postlarvae. J. Virol. Methods 74, 21–29.
Boonsanongchokying, C., Sang-oum, W., Sithigorngul, P., Sriurairatana, S., Flegel, T.W., 2006. Production of monoclonal antibodies to polyhedrin of monodon baculovirus (MBV) from shrimp. Sci. Asia 32, 371–376. Chaivisuthangkura, P., Tawilert, C., Tejangkura, T., Rukpratanporn, S., Longyant, S., Sithigorngul, W., Sithigorngu, P., 2008. Molecular isolation and characterization of a novel occlusion body protein gene from Penaeus monodon nucleopolyhedrovirus. Virology 381, 261–267. Chen, S.N., Chang, P.S., Kou, G.H., Lightner, D.V., 1989. Studies on virogenesis and cytopathology of Penaeus monodon baculovirus (MBV) in the giant tiger prawn (Penaeus monodon) and the red tail prawn (Penaeus penecillatus). Fish Pathol. 24, 89–100. Chen, S.N., Chang, P.S., Kou, G.H., 1992. Infection route and eradication of Penaeus monodon baculovirus (MBV) in larval giant tiger prawn Penaeus monodon. In: Fulks, W., Main, K.L. (Eds.), Diseases of Cultured Shrimp in Asia and United States. The Oceanic Institute, Honolulu, pp. 177–184. Fegan, D.F., Flegal, T.W., Sriurairatana, S., Waiakrutra, M., 1991. The occurrence, development and histopathology of monodon baculovirus in Penaeus monodon in Southern Thailand. Aquaculture 96, 205–217. Flegel, T.W., Nielsen, L., Thamavit, V., Kongtim, S., Prasharawipas, T., 2004. Presence of multiple viruses in non-diseased, cultivated shrimp at harvest. Aquaculture 240, 55–68. Johnson, P.T., Lightner, D.V., 1988. The rod-shaped nuclear viruses of crustaceans: gut-infecting species. Dis. Aquat. Organ. 4, 123–141. Liao, I.C., Su, M.S., Chang, C.F., 1992. Diseases of Penaeus monodon in Taiwan: a review from 1977 to 1991. In: Fulks, W., Main, K.L. (Eds.), Diseases of Cultured Penaeid Shrimp in Asia and the United States. Oceanic Institute, Honolulu, HI, pp. 113–137. Lightner, D.V., 1985. A review of the diseases of cultured penaeid shrimps and prawns with emphasis on recent discoveries and developments. Proceedings of the First International Conference on the Culture of Penaeid Prawns/Shrimps. Iloilo City, Philippines, pp. 79–103. Lightner, D.V., 1996. A Handbook of Shrimp Pathology and Diagnostic Procedures for Diseases of Cultured Penaeid Shrimp. World Aquaculture Society, Baton Rouge, LA. Lightner, D.V., Redman, R.M., 1981. A baculovirus-caused disease of the penaeid shrimp, Penaeus monodon. J. Invertebr. Pathol. 38, 299–302. Lin, C.K., 1989. Prawn culture in Taiwan, what went wrong? J. World Aquacult. Soc. 20, 19–20. Manisseri, M.K., Spann, K.M., Lester, R.J.G., 1999. Iodine as a disinfectant against monodon baculovirus (MBV). Asian Fish Sci. 12, 105–111. Mari, J., Bonami, J.R., Poulos, B., Lightner, D.V., 1993. Preliminary characterization and partial cloning of the genome of a baculovirus from Penaeus monodon (PmSNPV = MBV). Dis. Aquat. Org. 16, 207–215. Paynter, J.L., Vickers, J.E., Lester, R.J.G., 1992. Experimental transmission of Penaeus monodon type baculovirus (MBV). In: Shariff, M., Subasinghe, R.P., Arthur, J.R.
Fig. 4. Immunohistochemistry of the nauplius of MBV-infected Penaeus monodon using specific antibody against polyhedrin. The positive signals were detected in the body and appendages of the nauplius.
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(Eds.), Diseases in Asian aquaculture I. Fish Health Section, Asian Fisheries Society, Manila, pp. 97–110. Rajendran, K.V., Makesh, M., Karunasagar, I., 2012. Monodon baculovirus of shrimp. Indian J. Virol. 23, 149–160. Satidkanitkul, A., Sithigorngul, P., Sang-oum, W., Rukpratanporn, S., Sriurairatana, S., Withyachumnarnkul, B., Flegel, T., 2005. Synthetic peptide used to develop antibodies
for detection of polyhedrin from monodon baculovirus (MBV). Dis. Aquat. Org. 65, 79–84. Spann, K.M., Lester, R.J.G., Paynter, J.L., 1993. Efficiency of chlorine as a disinfectant against monodon baculovirus (MBV). Asian Fish Sci. 6, 295–301.
Contents lists available at ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aqua-online
An evidence on trans-ovarian transmission of Monodon baculovirus (MBV) infection in Penaeus monodon Duangkhaetita Kanjanasopa a, Pattira Pongtippatee a, Rapeepun Vanichviriyakit b, Somjai Wongtripop c, Padmaja Jayaprasad Pradeep b, Boonsirm Withyachumnarnkul b,c,⁎ a b c
Aquatic Animal Biotechnology Research Center, Faculty of Science and Industrial Technology, Prince of Songkla University, Surat Thani 84000, Thailand Department of Anatomy and Centex Shrimp, Chalerm Prakiat Building 4th Floor, Faculty of Science, Mahidol University, Rama 6 Rd, Bangkok 10400, Thailand Shrimp Genetic Improvement Center, Surat Thani 84110, Thailand
a r t i c l e
i n f o
Article history: Received 7 January 2015 Received in revised form 22 February 2015 Accepted 23 February 2015 Available online 7 March 2015 Keywords: Monodon baculovirus Trans-ovarian transmission Penaeus monodon Polyhedrin
a b s t r a c t Monodon baculovirus (MBV) causes slow growth of the shrimp being due to its infection in the hepatopancreas, the organ producing digestive enzymes and for nutrient storage. It has long been documented that the virus is transmitted from broodstock to offspring via contamination in rearing water by feces of the infected broodstock that contained sloughing-off damaged hepatopancreatic cells containing MBV. As a management practice, the washing of eggs and nauplii with disinfectant has been recommended to eliminate MBV that are attached to their surface and thus prevent the infection at the later development stage of the shrimp. However, we detected the MBV infection in postlarvae and juveniles of the black tiger shrimp Penaeus monodon, even after our repeated attempts to eliminate the virus by washing the eggs and nauplii with povidone iodine as disinfectant. Therefore we used MBV-infected broodstock to identify the cause of this problem by tracing the presence of MBV in hepatopancreas and ovary of the broodstock, eggs, nauplii and postlarvae, using histology with hematoxylin–eosin staining, polymerase chain reaction (PCR), in situ hybridization (ISH) and immunohistochemistry (IHC) specific for polyhedrin, a protein produced by MBV. We found all the ovaries collected from the broodstock, which were detected MBV-positive in the hepatopancreas by histology, PCR and ISH, were also PCR-positive. By ISH, positive signals were detected in the cell membrane, cytoplasm and nuclear membrane of the oocytes. The eggs and nauplii from the MBV-positive broodstock were also positive by PCR, in both “wash” and “no-wash” specimens. By ISH, positive signals were detected in ooplasm and subcuticular region of nauplii, as well as inside its body. Using IHC, positive signals were detected inside the body and appendage of the nauplius. Taken all these together, it is most likely that MBV could be vertically transmitted through trans-ovarian route. Hence, simply washing the eggs and nauplii with disinfectant may not be an adequate procedure to eliminate the MBV infection in offspring from MBV-infected broodstock. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Monodon baculovirus (MBV), an occluded double-stranded DNA virus of the family Baculoviridiae, could infect several species of penaeid shrimp, including the black tiger shrimp Penaeus monodon, from early to adult stages (Lightner and Redman, 1981; Lin, 1989; Liao et al., 1992; Rajendran et al., 2012). Infection with this virus usually does not cause shrimp mortality but high level of infection could result in slow growth of the shrimp and thus low production at harvest (Fegan et al., 1991; Flegel et al., 2004). The virus produces a matrix composed of polyhedrin protein surrounding its virion and thus is an occluded type. However, detailed study of MBV nucleocapsid and envelope structure has led a group of investigators to suggest that the virus should be grouped as a ⁎ Corresponding author at: Centex Shrimp, Faculty of Science, Mahidol University, 272 Rama 6 Rd, Bangkok 10400, Thailand. Tel.: +66 2 201 5871; fax: +66 2 354 7344. E-mail address: [email protected] (B. Withyachumnarnkul).
http://dx.doi.org/10.1016/j.aquaculture.2015.02.041 0044-8486/© 2015 Elsevier B.V. All rights reserved.
non-occluded one and renamed as singly envelope nuclear polyhydrosis virus from P. monodon or PnSNPV (Mari et al., 1993). The main target organ of infection by MBV in shrimp is hepatopancreas and its detection is easily observed by routine histology with hematoxylin–eosin (H&E) stain, in which the nuclei of the infected hepatopancreatic cells will reveal small multiple spherical acidophilic inclusions (Lightner and Redman, 1981). Quick diagnosis could be done by squash mounts of shrimp hepatopancreas and stained with malachite green, in which the occlusion bodies could be clearly observed (Lightner, 1985). In standard hatchery procedures, female broodstock are screened out for this virus prior to spawning by microscopic examination of fresh feces stained with malachite green (Lightner, 1996) or by polymerase chain reaction (PCR) specific for MBV. Early report suggests that MBV infection in the shrimp fry was transmitted from broodstock via feces contaminated with sloughing-off hepatopancreatic cells containing MBV occlusive bodies (Johnson and
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D. Kanjanasopa et al. / Aquaculture 443 (2015) 5–10
Lightner, 1988). The virus attach to the external surface of the eggs and nauplii. Since the nauplii have no fully developed mouth, the oral intake of the virus particles is rarely possible, but once they reach the zoeal stage and begin to have oral feed intake, the virus could reach the internal organs and slowly invade the hepatopancreas (Chen et al., 1989). Therefore, proper washing of eggs and nauplii thoroughly with disinfectants could free MBV infection at the later developmental stages of the fry. However, it was also reported that the disinfection procedure either requires high concentrations or a combination of more than one type of disinfectant (Chen et al., 1992; Spann et al., 1993; Manisseri et al., 1999). Although it is possible that the inclusion bodies function as a protector of MBV virions against the disinfectants (Mari et al., 1993), one cannot rule out another possibility that a fraction of MBV might be located intracellularly in the eggs and nauplii and thus escape the destruction by disinfectants. To investigate this possibility, we have traced the presence of gene and protein of polyhedrin, which is produced by MBV, in the ovaries of MBV-infected broodstock, and in eggs, nauplii and postlarvae (PL) generated by the broodstock, and the results reported herein. 2. Materials and methods 2.1. Sample collection Samples were collected from a commercial P. monodon hatchery. Feces of individual broodstocks were checked for the presence of MBV by PCR, and both MBV-negative and MBV-positive broodstocks were allowed to spawn individually in separate tanks. The eggs, nauplii, larvae and PLs were subsequently produced under standard hatchery production. The eggs and nauplii were divided into “wash” and “no-wash” groups. The “wash” group was those that were washed five times with povidone iodine (10 ppm) solution and three times with clean seawater, one-min each; and both disinfection and washing procedures were completely avoided for “no-wash” group. Samples collected from both MBV-negative and MBV-positive broodstocks and their respective fry at various stages were subjected to polyhedrin detections at both gene and protein levels, by in situ hybridization (ISH) and immunohistochemistry (IHC), respectively. Some samples were subjected to routine histology procedure with H&E staining and PCR detection for MBV as well (Table 1). Samples for routine histology, ISH and IHC were fixed in Davidson's fixatives and those for PCR were fixed in absolute ethanol. 2.2. Routine histology Samples were processed through paraffin sectioning with H&E staining (Lightner, 1996). Sections were observed particularly for the presence of multiple acidophilic occlusion bodies, a typical feature of Table 1 Sample collections and techniques performed. Number of determinations = 6. Sample Broodstock Feces Hepatopancreas Ovary Eggs (50 eggs/determinationa) “Wash” “No-wash” Nauplii (50 nauplii/determinationa) “Wash” “No-wash” PLs (20 PLs/determinationa) Derived from “wash” nauplii Derived from “no-wash” nauplii a
For PCR.
For PCR Histology, PCR, and ISH Histology, PCR, and ISH PCR and ISH PCR and ISH PCR, ISH and IHC PCR, ISH and IHC Histology and PCR Histology and PCR
MBV infection, in nuclei of the hepatopancreas of the broodstock and PLs, and ovaries of the broodstock. 2.3. PCR method All samples for PCR detection were kept in absolute ethanol and stored at −20 °C until use. DNA extraction was preceded by discarding absolute ethanol and the samples were grinded in 600 μl of lysis buffer containing 50 mM Tris–HCl, pH 9.0, 50 mM EDTA, 1.2 M NaCl and 0.2% Triton-X100 and heated at 95 °C for 15 min. After chloroform/isoamyl alcohol extraction, DNA was precipitated with absolute ethanol and its concentration and quality were measured by spectophotometric analysis at 260 and 280 nm. The amount used for each PCR determination was 50 ng DNA/μl. Nested PCR for detection of MBV was employed according to the method described by Belcher and Young (1998). Primers were designed based on sequence of the MBV polyhedrin gene (EU251062) (Chaivisuthangkura et al., 2008). Primary PCR and nested PCR amplification was performed with RBC Blue Taq Mastermix (RBC Bioscience). The primary PCR reaction was carried out in a 20 μl reaction mixture consisting of 100 ng DNA template, 10 μl of 2 × RBC Blue Taq Mastermix (final concentration: 10 mM KCl, 1.5 mM MgCl2 , 10 mM Tris–HCl (pH 8.3), 0.1 mg/ml BSA, 10 mM (NH4) 2 SO 4 , 0.2 mM dNTP mix, and 0.15 U/μl RBC Taq DNA polymerase), and 0.5 μM of pMBV-F (5′ATGTTCGACGATAGCATGAT-3′) and pMBV-R (5′-TTATTCATTTG TATGATGCG-3′). The cycle conditions for primary PCR amplification consist of: one cycle of 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 50 °C for 30 s, 72 °C for 90 s, and one cycle of 72 °C for 5 min. Nested PCR was carried out in a 25 μl reaction mixture consisting of 1 μl of the primary PCR reaction as the template for the nested PCR, 12.5 μl of 2 × RBC Blue Taq Mastermix (the final concentration was the same as above), and 0.5 μM of each internal (nested) primers as pMBV-NF(5′-CCACACAACATGCTTTGGAC-3′) and pMBV-NR (5′-GGCTAGACGCACACCAAGAT-3′). The second round amplification consists of: one cycle of 94 °C for 5 min; 25 cycles of 94 °C for 30 s, 50 °C for 30 s, 72 °C for 40 s, and one cycle of 72 °C for 5 min. Amplification products of the primary and nested PCR were analyzed by running at 1% of agarose gel and approximate sizes of the products were 1359 and 399 bp, respectively. 2.4. In situ hybridization All samples fixed with Davidson's fixative were kept at room temperature for 24 h before being transferred to 50% ethanol and stored at 4 °C until use. A labeled DNA probe to target the MBV polyhedrin gene was prepared using a commercial PCR DIG-labeling mix (Roche Molecular Biochemicals) according to the manufacturer's instructions. Nested PCR primers and protocol for the labeling reaction were produced as described above. DNA of MBV infected shrimp was used as template. The labeled probe used in a standard in situ hybridization assay method was adapted from the Genius™ Nonradioactive in situ Hybridization Application Manual by Boehringer Mannheim Biochemicals. The tissues were processed through paraffin sectioning and the paraffin-embedded tissues were placed on the tissue paper before placing them inside an oven (45–50 °C) for overnight. They were deparaffinized in fresh xylene 3 times for 5 min before re-hydration with series of alcohol followed by ethanol treatment at various concentrations in an ascending order; 100%, 95%, 80%, 70%, and 50% for 5 min each. After washing with fresh distilled water for 5 min, the sections were placed in TNE solution containing 50 mM Tris–HCl, 10 mM NaCl, 1 mM EDTA, (pH 7.0) for 5 min. Tissue sections were treated with proteinase K solution and incubated for 30 min at 37 °C in humid chamber. The sections were re-fixed with ice cold 0.4% formaldehyde solution for 5 min and immersed in distilled water for 5 min. Then, they were incubated at 37 °C for at least 10 min with pre-hybridization buffer. The buffer was carefully removed and hybridization buffer containing DIG-
D. Kanjanasopa et al. / Aquaculture 443 (2015) 5–10 Table 2 The presence of Monodon baculovirus in broodstock and nauplii as detected by polymerase chain reaction (PCR) and in situ hybridization (ISH) method. Hep, hepatopancreas; Ov, ovary. Batch no.
1 2 3 4 5 6
Broodstock Hep
Nauplius “Wash”
Ov
PCR
ISH PCR
ISH PCR
+ − + + − +
+ − + + + +
+ − + + − +
+ − − + − +
− − + − − −
PL “No-wash”
ISH PCR + − + + − +
− − + − − −
Derive from “wash” nauplii
Derive from “no-wash” nauplii
ISH
PCR
PCR
+ − + − − +
+ − + + − +
+ − + − − +
labeled probe was added. The sections were covered with cover slips and incubated at 42 °C overnight in humid chamber. The cover slips were removed and the sections were washed in washing solution. The
7
detection system was carried out by immunological method with NBT/BCIP system (Roche) according to the manufacturer's instructions. The nuclei were counter stained with 0.5% Bismarck brown Y. The tissues were then observed under light microscope. Negative controls were processed identically without the probe. 2.5. Immunohistochemistry The paraffin sections of the samples were de-paraffinized and rehydrated as described above. The sections were incubated with blocking reagent [4% bovine serum albumin and 10% normal goat serum in phosphate buffer saline containing 0.01% Tween 20 (PBST)] for 1 h and then with anti-polyhedrin antibody (MBV5-3H monoclonal antibody) (Satidkanitkul et al., 2005; Boonsanongchokying et al., 2006) at a dilution of 1:200 for 3 h at room temperature. The sections were washed 3 times with PBST and incubated with horseradish peroxidase conjugated goat anti-mouse IgG (Sigma-Aldrich) (1:500) for 1 h at room temperature. The sections were extensively washed with PBST and the antigen-antibody binding signal was developed using VECTOR
Fig. 1. Histology with H&E staining of samples of hepatopancreas (a) and ovary (b) of MBV-infected Penaeus monodon broodstock showing acidophilic multiple occlusion bodies in the nuclei of the hepatopancreas (a, arrow), but not in the ovary. By polymerase chain reaction, gel electrophoresis shows positive signal in both hepatopancreas (c, lanes 3, 5 and 6) and ovary (d, lanes 3, 6 and 9). M, DNA markers; P, positive control; Lane 1, negative control; Lanes 2–10, samples.
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NovaRED Peroxidase Substrate Kit (Vector laboratories). The negative control was performed in parallel but without the primary antibody. 3. Results and discussion 3.1. MBV transmission from broodstock to PLs Using PCR, the 4/6 of hepatopancreas and 3/6 of ovaries under the study were shown positive for MBV (Table 2). Three broodstock that had MBV-positive in the hepatopancreas also were MBV-positive in the ovaries. Using ISH, one more positive case (that was PCR-negative) was detected in the hepatopancreas, as well as in the ovary. Not all broodstock with MBV-positive ovaries by PCR method produced MBVpositive nauplii. By PCR detection, only 1/3 of the MBV-positive ovaries generated MBV-positive nauplii from “wash” specimens, while using ISH, 4/4 of the MBV-positive ovaries generated MBV-positive nauplii in “wash” specimen. In “no-wash” specimens, only 1/6 nauplius batches were MBV-positive by PCR, and the positive batch was not from the same broodstock that was MBV-positive in the ovary; however, by using ISH, 3/4 of broodstocks with MBV-positive generated MBVpositive nauplii. When nauplii, both “wash” and “no-wash” specimens, grew up to PLs, they were again determined for the presence of MBV by PCR and histology. The results from the PCR detection at this stage were correlated with those of nauplii detected by ISH. The above results suggested that (1) ISH was more sensitive than PCR in detecting MBV; (2) broodstock with MBV-positive in the ovary, by either PCR or ISH, generated MBV-positive nauplii; and (3) washing with povidone iodine in the eggs and nauplii could not completely eliminate MBV infection in the nauplii, as well as in PLs.
3.2. Localization of MBV By routine histology of hepatopancreas of broodstock and PLs, typical acidophilic multiple occlusion bodies in the nuclei were clearly observed; and the histological features were corresponded to the PCR results. In the ovaries, no occlusion bodies were observed in the histology of any specimen, despite 3/6 of the ovaries examined were MBVpositive by PCR. Fig. 1 shows histology with H&E staining of samples of hepatopancreas and ovary of MBV-infected broodstock, as well as a sample of gel electrophoresis for MBV detection by PCR, which shows a clear positive result in some ovarian samples that did not contain occlusion bodies. In the hepatopancreas, using ISH, the positive signals were detected in the nuclei of hepatopancreatic cells (Fig. 2b), while in the ovary, the positive signals were observed in the cell membrane, cytoplasm and nuclear membrane of oocytes (Fig. 2c). The size of the signals in the ovary was much smaller than those in the hepatopancreas. The reason behind this weaker signal detection of polyhedrin gene of MBV could be because of the lesser number of polyhedrin gene copies in the ovary than that in the hepatopancreas. The results also suggest that the absence of occlusion bodies in the ovary does not rule out MBV infection in the tissue. It is possible that in order to produce occlusion bodies, either sufficient number of MBV copies must exist or certain conditions in the host cells are required. Thus far, there has not been any report of MBV occlusion bodies in any other tissues besides hepatopancreas (Rajendran et al., 2012). There were no positive signals from the egg surface of both “wash” and “no-wash” eggs, when analyzed using ISH, however, positive signals were observed inside the ooplasm of the eggs (Fig. 3a), suggesting that MBV was inside the egg. Likewise, in nauplii, both “wash” and “no-
Fig. 2. In situ hybridization of the hepatopancreas and ovary of MBV-infected Penaeus monodon using specific probe for polyhedrin gene. The picture shows the hepatopancreas without probe (a) and with probe (b). Positive signals appear as black dots inside the nuclei of the hepatopancreatic cells. In the ovary (c), positive signals were detected as black dots on the cell membrane, inside the cytoplasm and on the nuclear membrane.
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Fig. 3. In situ hybridization of the egg (a) and nauplius (b) of MBV-infected Penaeus monodon using specific probe for polyhedrin gene. Positive signals were detected in the ooplasm and subcuticular epithelium, as well as inside the body of the nauplius.
wash” specimens, positive signals were observed underneath the subcuticular epithelium and inside the nauplius body (Fig. 3b). In addition, by IHC, positive signal was also observed inside the body of the nauplius (Fig. 4). Since the nauplius has not yet developed its mouth and the possible entry of MBV through oral route is less certain, hence the presence of MBV inside its body strongly suggest that MBV was derived from the egg and ovary of the broodstock. It is likely possible that the dose of povidone iodine used in this study might not be strong enough to destroy MBV on the surface of the eggs and nauplii, but the finding of positive expression of MBV polyhedrin inside the eggs and nauplii as revealed from ISH and IHC suggests that washing the eggs with disinfectant is not a reliable prophylactic measurement for eliminating MBV from the infected specimens. This study strongly suggests that MBV could be vertically transmitted via trans-ovarian route. However, the results of this study do not deny the fact that MBV infection could be transmitted horizontally as it has been proven previously that the transmission could occur by feeding MBV-contaminated hepatopancreas to PLs (Chen et al., 1992; Paynter et al., 1992). In practical point of view, this study revealed that disinfecting and washing eggs/nauplii with the purpose of “complete elimination” of MBV from their surface cannot be fully guaranteed. Hence, we strongly recommend revised strategies for the prevention of MBV in penaeid shrimp by eliminating all MBV-infected broodstock from the hatchery. Acknowledgments The study was supported by Prince of Songkla University, grant no. S&T550412S. References Belcher, C.R., Young, P.R., 1998. Colouremetric PCR-based detection of monodon baculovirus in whole Penaeus monodon postlarvae. J. Virol. Methods 74, 21–29.
Boonsanongchokying, C., Sang-oum, W., Sithigorngul, P., Sriurairatana, S., Flegel, T.W., 2006. Production of monoclonal antibodies to polyhedrin of monodon baculovirus (MBV) from shrimp. Sci. Asia 32, 371–376. Chaivisuthangkura, P., Tawilert, C., Tejangkura, T., Rukpratanporn, S., Longyant, S., Sithigorngul, W., Sithigorngu, P., 2008. Molecular isolation and characterization of a novel occlusion body protein gene from Penaeus monodon nucleopolyhedrovirus. Virology 381, 261–267. Chen, S.N., Chang, P.S., Kou, G.H., Lightner, D.V., 1989. Studies on virogenesis and cytopathology of Penaeus monodon baculovirus (MBV) in the giant tiger prawn (Penaeus monodon) and the red tail prawn (Penaeus penecillatus). Fish Pathol. 24, 89–100. Chen, S.N., Chang, P.S., Kou, G.H., 1992. Infection route and eradication of Penaeus monodon baculovirus (MBV) in larval giant tiger prawn Penaeus monodon. In: Fulks, W., Main, K.L. (Eds.), Diseases of Cultured Shrimp in Asia and United States. The Oceanic Institute, Honolulu, pp. 177–184. Fegan, D.F., Flegal, T.W., Sriurairatana, S., Waiakrutra, M., 1991. The occurrence, development and histopathology of monodon baculovirus in Penaeus monodon in Southern Thailand. Aquaculture 96, 205–217. Flegel, T.W., Nielsen, L., Thamavit, V., Kongtim, S., Prasharawipas, T., 2004. Presence of multiple viruses in non-diseased, cultivated shrimp at harvest. Aquaculture 240, 55–68. Johnson, P.T., Lightner, D.V., 1988. The rod-shaped nuclear viruses of crustaceans: gut-infecting species. Dis. Aquat. Organ. 4, 123–141. Liao, I.C., Su, M.S., Chang, C.F., 1992. Diseases of Penaeus monodon in Taiwan: a review from 1977 to 1991. In: Fulks, W., Main, K.L. (Eds.), Diseases of Cultured Penaeid Shrimp in Asia and the United States. Oceanic Institute, Honolulu, HI, pp. 113–137. Lightner, D.V., 1985. A review of the diseases of cultured penaeid shrimps and prawns with emphasis on recent discoveries and developments. Proceedings of the First International Conference on the Culture of Penaeid Prawns/Shrimps. Iloilo City, Philippines, pp. 79–103. Lightner, D.V., 1996. A Handbook of Shrimp Pathology and Diagnostic Procedures for Diseases of Cultured Penaeid Shrimp. World Aquaculture Society, Baton Rouge, LA. Lightner, D.V., Redman, R.M., 1981. A baculovirus-caused disease of the penaeid shrimp, Penaeus monodon. J. Invertebr. Pathol. 38, 299–302. Lin, C.K., 1989. Prawn culture in Taiwan, what went wrong? J. World Aquacult. Soc. 20, 19–20. Manisseri, M.K., Spann, K.M., Lester, R.J.G., 1999. Iodine as a disinfectant against monodon baculovirus (MBV). Asian Fish Sci. 12, 105–111. Mari, J., Bonami, J.R., Poulos, B., Lightner, D.V., 1993. Preliminary characterization and partial cloning of the genome of a baculovirus from Penaeus monodon (PmSNPV = MBV). Dis. Aquat. Org. 16, 207–215. Paynter, J.L., Vickers, J.E., Lester, R.J.G., 1992. Experimental transmission of Penaeus monodon type baculovirus (MBV). In: Shariff, M., Subasinghe, R.P., Arthur, J.R.
Fig. 4. Immunohistochemistry of the nauplius of MBV-infected Penaeus monodon using specific antibody against polyhedrin. The positive signals were detected in the body and appendages of the nauplius.
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(Eds.), Diseases in Asian aquaculture I. Fish Health Section, Asian Fisheries Society, Manila, pp. 97–110. Rajendran, K.V., Makesh, M., Karunasagar, I., 2012. Monodon baculovirus of shrimp. Indian J. Virol. 23, 149–160. Satidkanitkul, A., Sithigorngul, P., Sang-oum, W., Rukpratanporn, S., Sriurairatana, S., Withyachumnarnkul, B., Flegel, T., 2005. Synthetic peptide used to develop antibodies
for detection of polyhedrin from monodon baculovirus (MBV). Dis. Aquat. Org. 65, 79–84. Spann, K.M., Lester, R.J.G., Paynter, J.L., 1993. Efficiency of chlorine as a disinfectant against monodon baculovirus (MBV). Asian Fish Sci. 6, 295–301.