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.

Graduation date: 1993

Detection and treatment of bacterial kidney disease (BKD) was investigated. Experiments were conducted to evaluate the quantitative, fluorescent antibody technique (QFAT) that is used to detect, identify, and quantify both typical and 'bar form' Renibacterium salmoninarum cells. Smears of kidney tissue from naturally and artificially infected salmonids, both with and without chemotherapy, were quantitatively examined throughout the course of R. salmoninarum infections. Detection and quantification by QFAT has been reported to provide assessments of prevalence and severity of R. salmoninarum of individual fish. These assessments and the occurrence of 'bar forms' of R. salmoninarum have been used as an indication of recovery within a population. 'Bar forms' were observed in kidney tissue smears of fish that survived bacterial challenge when treated with erythromycin. The 'bar form' was also detected when rainbow trout were artificially infected with lower doses of live R . salmoninarum and in fish that were injected with irradiation-inactivated R. salmoninarum cells. By examining R. salmoninarum cultures in vitro by QFAT, it was determined that 'bar forms' did not occur on artificial media even when antibiotics were incorporated into the agar. When QFAT was compared to direct fluorescent antibody technique (DFAT) and quantitative enzyme linked immunosorbent assay (ELISA), it was determined that QFAT had similar sensitivity as ELISA but was more sensitive than DFAT. QFAT was also used to predict minimum mortality. Experiments were also conducted to evaluate drug regimes to treat both artificial and natural R. salmoninarum infections. Erythromycin was administered by intraperitoneal injection in different doses and at selected days post infection. Erythromycin decreased percent mortality and increased mean day to death, but did not completely eradicate R. salmoninarum from infected test animals. Sarafloxacin and erythromycin were incorporated into daily ration of artificially infected test animals. Contrary to erythromycin, sarafloxacin did not decrease mortality or increase mean day to death when tested in vivo against R. salmoninarum. A new drug, A-77143, was tested in vitro to determine if it was bactericidal and its minimum inhibitory concentration. When A- 77143 was compared to other antibiotics, it had a relatively low minimum inhibitory concentration and was shown to be bactericidal against the eight strains of R. salmoninarum tested. / Graduation date: 1993

The literature on Ichthyobodo necator is reviewed. The prevalence and intensity of Ichthyobodo infestations on farmed salmonids was investigated on three farms over a period of two years. The infestations were found to be markedly age dependent. Peak infestations and related mortalities occurred in the first eight weeks after first feeding. Both mortalities and infestations declined to zero shortly after this period with no chemotherapy. Ichthyobodo reappeared on 0+ and appeared for the first time on I+ fish after a drop of water temperatures to less than 10*C. Many of the 1+ fish had started to mature. It is suggested that some form of host defence mechanism operates which limits the Ichthyobodo infestations in farmed salmonids. The sequential pathology of Ichthyobodo infestations of the skin of 0+ and 1+ salmon and rainbow trout was studied. Areas of greatest shelter from water currents were found to be most commonly infested and no parasites were found attached to the epidermis on the head of the fish. The parasite caused hyperplasia of the malphigian cells and exhaustion of the goblet cells below infestations, followed by spongiosis of the underlying epidermis. The epidermal plaque then sloughed off leaving a single layer of cells attached to the basement membrane. Cell kinetic studies showed that Ichthyobodo caused the cells immediately below infestations to divide, a markedly different pattern from that of normal teleost epidermal cell proliferation. The possibility that the parasite secretes some form of digestive enzyme is postulated. In areas where sloughing had occurred, the remaining malphigian cells were seen to be in the process of division. Various endocrinological aspects of Ichthyobodo infestations were investigated. Three corticosteroids and one androgen were injected or implanted into 1 year old rainbow trout. Implantations of hydrocortisone led to very heavy ichthyobodo infestations. Radio immune assays showed that the level of cortisol and testosterone in the serum of implanted fish was similar to that which would occur when salmonids mature. There appears to be a clear link between cortisol levels in the serum and Ichthyobodo infestation. The host response to Ichthyobodo is discussed and it is concluded that cortisol may suppress the host's defence mechanism to Ichthyobodo.

Graduation date: 1997

Little is known about the virulence factors of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. The predominant protein produced by R. salmoninarum in broth culture or during infection is a 57/58 kDa protein (p57) which is associated with strain virulence. In this study monoclonal antibodies (MAbs) to p57 were developed and used as tools to antigenically characterize and quantify the protein. Monoclonal antibodies 4D3 and 2G5 recognize p57 and appear to be species specific as they did not cross-react with proteins produced by bacterial species within the genera Streptococcus, Carnobacterium, Vibrio and Aeromonas, or with fish serum proteins. Further, these MAbs recognize conserved epitopes on p57 shared by 10 isolates from geographically diverse areas. In vitro activities attributed to p57 include the suppression of antibody production, and the agglutination of rabbit erythrocytes and salmonid spermatocytes. We described a novel in vitro agglutinating activity of p57 toward salmonid leukocytes that was inhibited by two of a panel of eight MAbs. The location of the putative epitopes recognized by the MAbs were determined by two-dimensional electrophoresis and Western blotting of proteolytic breakdown fragments of p57. Amino acid sequencing of several of the fragments suggested that the antibodies which inhibit agglutinating activity bind proximal to the amino terminus of the protein. To investigate the mechanism of leukocyte agglutination, p57 was purified to near homogeneity using anion-exchange and size-exclusion fastpressure liquid chromatography. P57 eluted as a protein monomer and retained leukoagglutinating activity. In addition, results of antibody-capture, enzyme-linked immunosorbent assays suggest that a monomer exists in culture supernatant and infected fish tissue. Antigenic analysis with MAbs has also been useful for developing immunoassays for detecting and quantifying p57 levels in vivo. Using a quantitative ELISA, the prevalence of salmon with antigen levels above 3 ng/ml of kidney homogenate varied from 12.8 to 36.6% in 740 adult spawning chinook salmon returning to an Oregon hatchery from 1989 to 1991. A rapid, semi-quantitative, Field ELISA was also developed for use under hatchery conditions, in addition to a sensitive chemiluminescent Western blot protocol for confirming ELISA positive samples. / Graduation date: 1992