Investigation of the microbial flora and immune function of abalone as indicators associated with Summer Mortality Syndrome and development of possible preventatives

Abstract

Abalone summer mortality syndrome (ASMS) broadly defines a mass mortality event in abalone that occurs when water temperature is above 23˚C. It is proposed that heat stress affects the metabolism of the abalone making them more susceptible to opportunistic pathogens, such as pathogenic Vibrio harveyi strains. In order to provide a better understanding of ASMS and provide important information for the Southern Australian greenlip abalone (Haliotis laevigata) industry, the incidence of bacteria-related cases diagnosed from abalone mortalities around Australia and New Zealand between 2011 and 2017 was investigated. Results from diagnostic laboratory data indicated that there were no major bacterial disease outbreaks in abalone between 2011 and 2017 in the investigated area, but it was noted that surveillance of abalone health was only actively undertaken in Tasmania, with a focus on abalone viral ganglioneuritis. The study included investigation of the correlations between abalone meat weight, footprint size and shell characteristics (including shell length, width and depth). Two potential management systems (including grading) were proposed based on the observed physical correlation between abalone meat weight and its footprint size. The activity of bacteriophages (phages) against pathogenic Vibrio. harveyi strains was assessed and characterised, and phage therapy was assessed in vitro and in vivo. Four phages active against V. harveyi strain MO10 and three phages active against strain M071 were identified and characterised. The results of phage in vitro testing against MO10 provided the potential dose rate for the in vivo treatment experiments. The survival of V. harveyi infected abalone increased to 70% compared to 0% in controls in the in vivo tests. In in vitro tests, three phages that infected V harveyi MO71 were trialled either individually or mixed at 1:1:1 volume ratio at temperatures 25˚C, 28˚C and 31˚C, and bacterial optical density curves were generated to compare the effectiveness of single phage treatments and the cocktail. The results indicated that individual phages or the cocktail were more effective at 28˚C and 31˚C. And treatments with higher concentrations of phage in general showed better efficacy. Interestingly, the cocktail was not the most effective treatment when compared to use of a single phage. The microbial flora of the left kidney and the stomach were sequenced in both summer and winter in 3-year-old adult abalone from both surgically excised tissue and tissue samples aspirated by needle and syringe. The V3-V4 regions of 16S rRNA were sequenced to profile the microbial flora of the left kidney and stomach. The results demonstrated the microbial differences between these two organs. In addition, there were seasonal differences in the microflora of the targeted organs. Collection of needle aspiration samples was abandoned as a number of samples did not meet the quality control required for sequencing and because of the relatively low alpha and beta diversities in the sequencing results. Sequencing results indicated the left kidney harbours diverse bacteria and is potentially indicative of the abalone physiological status, offering an alternative to the stomach for microbial profiling of abalone. The seasonal differences in abalone stomach microbiome structure provide information for probiotic development and health management for the abalone industry. It would be beneficial for both industry and researchers to develop a surveillance program allowing the development of a database of veterinary diagnostic reports including identified infectious agents. Potential industrial procedures could be developed based on such information with the aim of reducing potential stress during grading and increasing the effectiveness of on farm management. Phage therapy is recommended for disease treatment but not as prevention considering the cost and effectiveness of the treatment. Future research should focus on administration routes of the phage therapy as well as the mechanisms behind the dynamic relationships of phage, bacterium, the abalone and environmental temperatures. To decrease the mortality and reduce the welfare impact on the abalone, an alternative sample collection method needs to be developed for abalone sequencing-related work. More frequent profiling can provide a better understanding of the dynamics of abalone microbial communities, and sequencing at different stages of development of abalone can provide more information for health management and probiotic development in the industry. Introduction to thesis: Summer mortality is common in aquaculture especially in shellfish and crustacea (Lemonnier et al. 2006, Xiao et al. 2005, Yu et al. 2010, Oliveira et al. 2015). The definition of abalone summer mortality syndrome (ASMS) given by Stone et al. (2014) is a disease caused by an interaction between biotic and abiotic environmental factors when the water temperature is above 22 ⁰C, as this results in negative impacts on the health, growth and mortality of mature cultured abalone in southern Australia. This definition was derived from the concept of summer mortality in oyster farming (Samain 2011, Mortensen et al. 2016). As abalone farming depends on intensive land-based culture systems and the inherent characteristic of univalve molluscs is that they take a considerable resource to support them to market, these mortality events result in dramatic economic loss. To date, there is no agreed definition of ASMS. It is probable that this syndrome is complex, and in the past, it has not been monitored by any indicators other than mortality. The aims of this project are: to investigate the role of pathogenic bacteria in mass mortality events in the abalone industry during the last five years; to innovate grading methods for the abalone industry to reduce stress and potential mortality during the process; to develop bacteriophage therapy for the abalone industry; to investigate the microbiota of abalone left kidney and stomach in both summer and winter using different methods. Based on the results of the above mentioned studies, establishment of a better understanding of the relationship of stress and microbial flora may improve our knowledge of ASMS and potential responses or solutions can be suggested. The thesis was divided into seven chapters. Chapter one is a review of published literature and revisits the current methods for assessment of abalone health with a focus on abalone immunology and microbial profiling, and identifies gaps of knowledge in these areas. Chapter two reports a survey of diagnostic laboratories within Australia and New Zealand between 2011 and 2017 which sought information on the role of bacterial agents associated with abalone mortalities that had been investigated. Chapter three introduces two potential grading systems that are based on developing an equation to estimate the body weight of the abalone from physical parameters. Chapter four and five describe the development of bacteriophage therapy techniques for the abalone industry using infection with V. harveyi as the bacterial model. Chapter six describes a microbial sequencing study of adult abalone left kidney and stomach in both summer and winter using tissue aspiration and excision. Chapter seven is a discussion of findings and proposals for future research in the area of ASMS.