Piscirickettsia salmonis : characterisation, infection and immune response in salmonid fish

The pathogen Piscirickettsia salmonis, has been isolated from all species of salmonid and has been found in Chile, Canada, Ireland, Norway and Scotland. Rickettsia-like organisms from European sea bass (Dicentrarchus labrax) were found to share common antigens with the P. salmonis type-strain, LF-89 using the indirect fluorescent antibody test (IFAT) and immunohistochemistry (IHC). In addition, the DNA sequences of the 16S rDNA and 16S-23S internal transcribed spacer region (ITS) were compared with those published for P. salmonis strains and showed that the sea bass piscirickettsia-like organism (SBPLO) was another strain of P. salmonis, closely related to the salmonid pathogens. The ability of P. salmonis to survive and replicate within head kidney (HK) macrophages of rainbow trout infected in vitro was demonstrated using transmission electron microscopy (TEM) at various times post-infection (p.i.). However, macrophages derived from fish vaccinated against P. salmonis appeared to clear in vitro infection more rapidly than macrophages from naive fish. Polymerisation of filamentous actin within the cytoplasm of the host cell is used by some mammalian rickettsiae to achieve intercellular spread by actin-based motility (ABM). Both TEM and confocal microscopy were used to investigate possible actin tail formation by P. salmonis. No evidence of tail formation was found. Respiratory burst (RB) by P. salmonis was measured following exposure of rainbow trout HK macrophages to the organisms in vitro. Because of background stimulation of the RB by growth media and debris from the CHSE-214 cells used to culture P. salmonis, it was not possible to detect any effect of the pathogen on the burst. Schering Plough Aquaculture has developed a recombinant vaccine against P. salmonis. The ability of the vaccine to elicit a memory response against P. salmonis was investigated by measuring three different immune responses: a) the expression of iNOS was measured by reverse transcription polymerase chain reaction (RT-PCR) to detect mRNA levels or by the Greiss reaction to quantify the end-products of nitric oxide metabolism in the serum. Increased iNOS expression was not detected in rainbow trout kidney or serum following vaccination/challenge with P. salmonis. However, iNOS expression was detected in gill tissue from naive trout which suggests that expression may be constitutive in this tissue. b) the production of macrophage activating factor (MAF) by lymphocytes from vaccinated trout, following stimulation in vitro with P. salmonis, was measured by the ability of supernatants from these cells to prime elevated RB in naive macrophages. No difference in priming ability between supernatants from vaccinated and non-vaccinated fish was detected. However, macrophages among the immune leukocytes used to produce the MAF supernatants did exhibit elevated RB compared with macrophages from non-immune fish, suggesting that vaccination had produced a population of lymphocytes capable of priming activation of macrophages. c) by screening individual sera concurrently against the rickettsial and CHSE antigen preparations, the antibody response to P. salmonis could be detected specifically and was found to increase significantly in immunised fish by 6 weeks post-vaccination. Specificity of the response was demonstrated by screening the sera against Aeromonas salmonicida.EThOS - Electronic Theses Online ServiceAquaculture Vaccines Ltd : University of Stirling CASE studentshipGBUnited Kingdo