Author: Toan Vinh Dang
Dang, Toan Vinh, 2012 Antiviral immune responses in abalone and influence of potential abiotic and biotic factors, Flinders University, School of Biological Sciences
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Abalone (Haliotidae) are marine gastropod mollusks and important aquaculture species worldwide. Unfortunately, severe mortality of abalone caused by a herpesvirus (AbHV) has been reported in Australia. The manifestation of disease involves an interaction between virus, environment and abalone immunity (Chapter 1). Therefore, this research aimed to investigate the presence of antiviral activity in abalone Haliotis laevigata (greenlip), H. rubra (blacklip) and their hybrid. Due to the lack of molluscan cell lines for culturing AbHV, antiviral activity of abalone was assessed against a similar neurotropic herpesvirus, herpes simplex virus type 1 (HSV-1) using the plaque assay. Assessment of antiviral activity was complemented with other immune assays, measuring total haemocyte count (THC), phagocytosis, phenoloxidase activity, respiratory burst and antibacterial activity against Vibrio spp. to provide an overall view of immune status in abalone after exposure to various biotic and environmental factors. A number of abalone organs were screened for anti-HSV-1 activity, but only the haemolymph (20%, v/v) and the lipophilic extract of digestive gland (3,000 [mu]g ml-1) were found to substantially decrease the number and size of virus plaques (Chapter 2). Haemolymph inhibits viral infection at an early stage (e.g. viral entry) whereas the antiviral effect of the lipophilic extract is greatest when added one hour after infection (e.g. the intracellular stage of viral infection). There was considerable variation in the levels of antiviral and antibacterial activity in the haemolymph among abalone within the same aquaculture family lines and natural populations in different geographic locations (Chapter 3). Antiviral and antibacterial activity increased slightly with an increase in shell length. However, there was no significant effect of gender or spawning status on antiviral or antibacterial status. Concomitant with strong antiviral activity against HSV-1 in a lipophilic extract of Ulva lactuca and Spyridia filamentosa, higher antiviral activity was detected in the digestive gland lipid extract of abalone fed Ulva lactuca (64.2% at 650[mu]g ml-1) or Spyridia filamentosa (69.51%) compared to abalone fed pellets (47.42%) or pellets supplemented with Arthrospira maxima (46.3%) or Dunaliella salina (46%) (Chapter 4). There was no influence of diet on the humoral antiviral activity, indicating antiviral factors in the haemolymph are likely to be innately biosynthesized by the abalone. Sampling of wild-caught H. rubra showed a significant correlation between temperature and antiviral or antibacterial activity, with higher activity in summer than in winter months (Chapter 5). However, antibacterial activity was compromised in favour of antiviral activity as the water temperatures peaked in summer. A controlled laboratory experiment with water temperature raised from 18 to 21 or 24 [mu]C showed that THC and SO increased at day 1 and then dropped back to control levels by days 3 and 7. By comparison, the humoral immune parameters showed a delayed response with antibacterial and antiviral activity significantly increasing on days 3 and 7, respectively. Consistent with the field study, antibacterial activity became significantly depressed after prolonged exposure to elevated temperatures. Consequently, abalone may have more resilience to viruses than bacterial pathogens under conditions of elevated temperature. Experimental transmission trials were undertaken using an immersion model to study the abalone immune response to infection with AbHV (Chapter 6). The infection status of abalone was confirmed by real-time PCR. THC decreased by 38.8% in moribund abalone, but increased by 42.6 and 13.6% in apparently healthy abalone that were PCR-negative and PCR-positive for AbHV, respectively, in comparison to the non-infected group. The level of SO decreased in abalone confirmed as PCR-positive for AbHV, by 30.8% in moribund abalone and by 7.2% in apparently healthy abalone. However, for apparently healthy abalone that were PCR-negative after viral challenge, SO significantly increased, by 59.3%, in comparison to uninfected controls. These results suggest that THC and SO provide potential immune markers for AbHV infection status. In conclusion, abalone have at least two antiviral compounds with different modes of action against viral infection. Humoral antiviral factors appear to be constitutively produced and are influenced by high temperature but not by diet or infection status. Further investigation is required to establish whether the individual variability in antimicrobial activity is heritable in breeding programs and whether higher activity confers greater resistance to disease.
Keywords: abalone,immune responses,antimicrobial activity,herpesvirus infection
Subject: Biological Sciences thesis
Thesis type: Doctor of Philosophy
School: School of Biological Sciences
Supervisor: Dr Peter Speck