Standardization of diagnostic methods for monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV): Establishment of monoclonal antibodies (MAbs) against MBV and HPV
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2005-03Page views
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Abstract
Monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV) remain as two of the serious pathogens that infect black tiger shrimp (Penaeus monodon) in the Philippines. It is, therefore, necessary to prevent the spread and occurrence of such pathogens, especially in shrimp hatcheries where postlarvae (PLs) stocked in culture ponds are usually obtained. One effective means is to establish rapid and reliable diagnostic techniques for these viruses.
This research was undertaken mainly to develop monoclonal antibodies (MAbs) against MBV and HPV. Consequently, these MAbs will be used as primary reagents to establish an appropriate immunologically-based detection method for MBV and HPV. Immunologically-based diagnostic methods have proven to be rapid, sensitive and quite specific. Furthermore, they could be performed in the field using very simple set-up.
Four main studies were conducted for this research. The first involves the development of infection model of HPV in P. monodon. Series of experiments showed that HPV could be horizontally-transmitted in postlarval P. monodon through the oral route. This allowed the availability of infected materials to be used as immunogen and for screening of antibodies. The infection model also provided a method by which the pathogen could be further studied, including its control in hatchery conditions. The second study describes the development of MAbs against HPV and MBV, including the optimization of an immunofluourescent antibody test (IFAT), as a screening technique. The third study involves the production of polyclonal antibodies (PAbs) against MBV and HPV wherein IFAT, immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA) were optimized and used to screen the antibodies as to their usefulness in MBV and HPV diagnosis. The fourth set of experiments were undertaken to determine the efficiency of chlorine as a disinfectant in inhibiting HPV infection in P. monodon PLs. The results of each study are discussed in this report.
Suggested Citation
Catap, E. S., & de la Peña, L. D. (2005). Standardization of diagnostic methods for monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV): Establishment of monoclonal antibodies (MAbs) against MBV and HPV. In K. Nagasawa (Ed.), Recent Advances in Diagnosis and Prevention of Fish and Shrimp Diseases in Southeast Asia (pp. 27–28). Tigbauan, Iloilo, Philippines: Aquaculture Department, Southeast Asian Fisheries Development Center.
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9718511732Related items
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Production of polyclonal antibodies (PAbs) against monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV)
(Aquaculture Department, Southeast Asian Fisheries Development Center, 2005-03)Monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV) are two of the viral pathogens, which infect penaeid shrimps in the Philippines. Polyclonal antibodies (PAbs) to MBV and HPV were raised by immunization of rabbits with the purified virions. The antisera produced were assessed using immunofluorescence antibody test (IFAT), indirect enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC). IFAT showed intense fluorescent reactions in impression smears and paraffin-embedded sections of MBV-infected P. monodon postlarvae (PLs) but not in smears and/or sections infected with HPV or WSSV. Similarly, IHC tests showed positive coloration in MBV-infected cells but not in sections with either HPV or WSSV. Indirect ELISA test revealed that the PAbs could detect 4 ng (rabbit 1) and 100 ng (rabbit 2) of purified MBV. However, testing of crude shrimp homogenates by indirect ELISA indicated high level cross-reactions (gills of WSSV-infected adult P. monodon, head portions of HPV- and MBV-infected PLs and non-infected PLs). Screening of anti-HPV serum likewise showed intense immunofluorescence and positive red brownish coloration in HPV-infected sections. However, there were also strong cross-reactions observed in sections of hepatopancreas with MBV infection. Overall, the MBV PAbs developed have potential application in the detection of the virus. Further purification of the PAbs is required to minimize background and/or cross-reactions. -
Development of monoclonal antibody (MAb) against monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV)
(Aquaculture Department, Southeast Asian Fisheries Development Center, 2005-03)Antibody-based detection methods are relatively simple and rapid, aside from being specific and sensitive. Thus, there is a necessity to develop such techniques for early and rapid diagnosis of viral pathogens such as MBV and HPV. In this study, the development of monoclonal antibodies (MAbs) against MBV and HPV employing the hybridoma technology was initiated. Spleen cells from Balb/C mice, immunized with either semi-purified HPV suspension or purified MBV virions were fused with Sp2/0 myeloma cells to produce hybridoma cells. Supernatants from cells which showed good viability and growth were harvested and screened for the presence of antibodies against the two viruses. An indirect immunofluorescence antibody test (IFAT) was optimized to screen these supernatants. Results showed that 84 of the supernatants raised against HPV showed intense immunofluorescent reaction on either hepatopancreas impression smears or paraffin-embedded tissue. Of the 9 MBV supernatants, only 2 showed intense reaction and one showed very weak fluorescence. These results suggest that antibodies to detect these two pathogens could be derived from the hybridoma cells produced in this study. -
Bacteria and toxin isolated from the dinoflagellate Pyrodinium bahamense var. compressum and production of monoclonal antibodies and diagnostic kits to monitor red tide and toxic mussels
(Bureau of Agricultural Research, Department of Agriculture, 2007)Six bacterial isolates obtained from the red tide dinoflagellate Pyrodinium bahamense var. compressum were found to be toxic. The most toxic isolate MM-11 was cultured, characterized, and identified to be Micrococcus luteus. MM-11 and M. luteus had similar DNA bands on agarose gel, and contained 70.0–75.5% mole G+C. Several Micrococcus species were isolated from pure culture and field samples of Pyrodinium and from red tide affected mussels. MM-11 and the other Micrococcus isolates tested positive for saxitoxin. MM-11 was grown on seawater agar; peak cell density of 1.36 x 1010 cells/ml occurred after 3 days of incubation. Toxin production was directly proportional to cell density. The crude toxin from the optimized culture of MM-11 resulted in death of mice in only 1.8–2.4 min, equivalent to a toxicity of 5.9–13.4 mouse units. MM-11 was inoculated into healthy mussels and yielded bacterial isolates that had characteristics of MM-11, and extracts of toxin similar to MM-11 toxin. Mice injected with extracts from the inoculated mussels showed symptoms of paralytic shellfish poisoning (dyspnea 12–15 min after injection), but did not die. Partially purified extracts from red tide affected mussels killed mice in 3.4 min, equivalent to a toxicity of 3.4 mouse units. Addition of 5, 25 and 50% coconut milk to this toxin extract reduced the toxicity to only 34%, 29%, and 25% of that without coconut milk. The ELISA test similarly showed reduction of saxitoxin concentration from 4.78 g toxin/g at 5% added coconut milk to 3.62 g toxin/g at 50% added coconut milk. PSP toxins were extracted from bacteria and red tide affected mussels. The 24 purified extracts of MM-11 toxin were shown by mouse bioassay to have concentrations from 0.6 to 71.6 μg toxin/g bacteria. Green mussels sampled from Bataan and Zambales during incidence of red tides from 1994 to 1998 contained lower amounts of toxin per unit weight than the bacterial extracts. Analysis of the MM-11 toxin by HPLC-fluorometry showed two fractions similar to those of standard gonyautoxin 1 and gonyautoxin 3.
