Fungal diseases

The incidence of ichthyophoniosis in groupers (Epinephelus) has been reported in Plectropomus sp. in Singapore and Cromileptes altivelis in Indonesia. It has also been known to infect at least 80 other species of teleost fish from marine, estuarine and freshwater habitats in both temperate and tropical regions (e.g., rainbow trout, yellowtail, mackerel, herring, flounder and cod). The etiologic agent of this disease is reportedly of uncertain taxonomic affinity but is often described as a fungus

Development of a method for reproducing epizootic ulcerative syndrome using controlled doses of Aphanomyces invadans in species with different salinity requirements

Lesions typical of epizootic ulcerative syndrome (EUS) were induced in three-spot gourami, Trichogaster trichopterus, and sand whiting, Sillago ciliata, injected intramuscularly with controlled doses of Aphanomyces invadans zoospores, the fungal pathogen associated with the disease. Both species of fish exhibited chronic granulomatous response and inflammatory cells, predominantly macrophages and lymphocytes, infiltrated the muscle and skin tissues, at days 6–8 post-inoculation of 65 to 85 spores/fish. Based on the comparative granuloma counts and percentage of cellular infiltration in a sampled lesion area using image analysis, it was shown that the three-spot gouramis mounted a more vigorous response than the sand whiting. It was also observed that lesions in three-spot gouramis exhibited early signs of resolution than those in sand whiting. However, fish mortality was greater in EUS-affected three-spot gourami than in EUS-affected sand whiting. With this technique, we were able to describe and compare the sequential histopathology of EUS lesions in a freshwater (three-spot gourami) and an estuarine (sand whiting) fish species.The authors are grateful to the Australian Centre for International Agricultural Research for the financial assistance; the sand whiting were provided by the Bribie Island Aquaculture Research Centre, DPI Queensland while the A. invadans (24 P) isolate, originally isolated by Ms. Annette Thomas, was provided by Dr. R.B. Callinan; Mr. Craig Thomas provided technical assistance with the rearing of juvenile sand whiting

Experimental transmission of hepatopancreatic parvovirus (HPV) infection in Penaeus monodon postlarvae

Hepatopancreatic parvovirus (HPV) infection in penaeid shrimps was first reported in various countries of the Asia-Pacific region in mid-1980. The virus affects the hepatopancreas of postlarvae and juveniles, usually leading to slow growth and mortality during the early stage of culture. At present, there is no established experimental model of infection in Penaeus monodon, a susceptible species, since there has not been any report of successful HPV transmission under laboratory conditions. Therefore, experiments were undertaken to induce HPV infection by feeding P. monodon postlarvae (PL) with virusinfected PL. Postlarval P. monodon (PL-16), initially examined to be free from HPV, were found HPV-positive 24 hours after they were fed with the infected material. Percentage of infection was from 30% (day 1) to 100% (day 7) based on the examination of wet mounts of hepatopancreas (squashed tissue) stained with malachite green and through histopathology. This is the first report of a successful horizontal transmission of HPV in P. monodon PL. This infection model could be used to study the pathogen further and would permit controlled experiments to be undertaken in order to identify methods of prevention and control

Production of polyclonal antibodies (PAbs) against monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV)

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

Studies on the experimental transmission model of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV) in shrimp (Penaeus monodon)

Series of experiments on the transmission of monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV) in Penaeus monodon were undertaken. Feeding of MBV-infected carcasses to experimental shrimps resulted to 100% infection at day 4 post-infection as determined by wet-mount technique and histopathology of hepatopancreas. Oral route of infection was also efficient in inducing HPV infection in postlarval P. monodon. Percentage of infection was 100% at 5-7 days post-infection which was also determined using the wet-mount technique, originally developed for MBV, and through histopathology. Further studies on the transmission of HPV showed that age of shrimps is an important factor in its pathogenicity. Results showed that HPV could be transmitted efficiently in postlarval P. monodon, but less efficient in juveniles. No infection was induced among the adult shrimp samples. It was also shown that the oral route of infection is more efficient in transmitting HPV rather than water-borne inclusion bodies. This is the first report of horizontal transmission of HPV in postlarval P. monodon in the Philippines

Histology as a tool in disease diagnosis

Histology is an important tool in fish disease diagnosis as it affords the comparison of normal structures or morphology of tissues against those from diseased fish. However, correct diagnosis and confirmation of changes associated with diseases require proper specimen processing and some degree of expertise in histopathology. The four basic types of tissues are: epithelial, connective, muscular and nervous. An organ is usually a combination of these four tissue types. It is important to remember that the histology or structure of an organ is always related to the function it performs

Chlorine disinfection against hepatopancreatic parvovirus (HPV) infection in shrimp (Penaeus monodon) larvae

Experiments were undertaken to determine the efficacy of chlorine as a disinfectant in inhibiting HPV infection among P. monodon postlarvae (PLs). Based on histopathological examination, 50 ppm and 200 ppm active chlorine at two different exposure durations (12 and 24 hr), failed to inhibit the transmission of HPV in the experimental PLs. Higher chlorine concentrations, 500 ppm and 1,000 ppm for 24 hr exposure, decreased percentage of HPV infection, but failed to completely inhibit the transmission of the virus in PLs. It is recommended that other disinfecting agents be tested as HPV appears to be resistant to chlorine. Also, the experimental design should be modified in order to actually simulate existence of HPV as an infectious agent in hatchery conditions

Development of monoclonal antibody (MAb) against monodon baculovirus (MBV) and hepatopancreatic parvovirus (HPV)

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