Vaccination of Atlantic salmon (Salmo salar) against marine flexibacteriosis

Tenacibaculum maritimum (formerly Flexibacter maritimus) is a well known pathogen in a number of cultured fish species worldwide. It is a marine bacterium that causes necrotic lesions on the body, head, fins and gills, with erosive lesions on the external surface as the prominent clinical sign. A large outbreak of the disease occurred in Tasmania over 15 years ago causing significant mortalities. Management strategies have reduced the incidence of outbreaks, however smaller outbreaks continue to occur. There is limited information on the biological factors that could initiate further outbreaks of the disease and patterns of infection are variable. Chemotherapeutants such as antibiotics are undesirable for use with problems of bacterial resistance, residence times in tissues and effects on other flora and fauna in the surrounding marine environment. Therefore, development of a safe and effective vaccine against T. maritimum is the focus of this research. To characterise and to understand the similarities and differences between T. maritimum isolates, several physical characterisation tests were carried out. The Tasmanian isolates of T. maritimum appeared relatively homogeneous physically, but were antigentically different. All isolates were hydrophobic and produce a variety of extracellular product (ECP) profiles. There were two isolates that stood out in all assays (89/4747 and 01/0356-7) and were different from the other isolates. Based on their physical characteristics, three isolates (89/4747, 89/4762, 00/3280) were chosen for further investigation in pathogenicity trials. Pathogenicity trials were carried out to test the effects of culture media, virulence of strains, confirm Koch' s postulates and develop a model of infection so that the efficacy of the experimental vaccines could be established. Culture media did not affect the pathogenicity of T. maritimum, but the presence of aeration within the culture broth revealed a dose effect caused by the cohesive nature of the bacteria. Isolate 89/4747 was found to be non-pathogenic, while the other isolates (89/4762, 00/3280) were highly pathogenic causing 100% mortalities within 3 days. The more moderate isolate 89/4762 was chosen to develop the model of infection and to be used in the subsequent vaccination trial. A model of infection using immersion as the challenge method was established for Atlantic salmon (Salmo salar). An `LD_60` of approximately `1` x \(10^6 cells/mL\) was calculated for the experimental period and this was the dose used for the vaccination trial. The possible role of extracellular products (ECP) in the pathogenicity of T. maritimum and its toxicity was investigated in viva by injecting different doses of ECP into the peritoneal cavity of Atlantic salmon. The ECP was found to be toxic and caused internal necrosis and haemorrhaging. An `LD_50` calculated for the experimental period was 125 μg/fish. A vaccination trial was conducted using a formalin de-activated vaccine and the vaccine plus an adjuvant (Freund's incomplete). Some protection was provided with the vaccine alone (RPS = 22.0-27.7%) but significant protection was achieved with the vaccine plus adjuvant (RPS = 78.0- 79.6%). The adjuvant caused pigmentation and inflammation in the pyloric caeca of the fish. For future work it is suggested that further characterisation of isolates are required, that toxins such as extracellular products (ECP) and lipopolysaccharide (LPS) be investigated in more depth and that duration of protection for the vaccine and alternative adjuvants be examined. In the short-term, the vaccine preparation from this study may be useful to the salmonid aquaculture industry in Tasmania, Australia