Use of Arthrobacter davidanieli as a live vaccine against Renibacterium salmoninarum and Piscirickettsia salmonis in salmonids.

Arthrobacter davidanieli (proposed species nomenclature) is a non-pathogenic Gram-variable bacterium related to, but taxonomically distinct from, Renibacterium salmoninarum, the aetiological agent of bacterial kidney disease (BKD). We have demonstrated that vaccination with live A. davidanieli is effective against BKD in Atlantic salmon (Salmo salar) showing above 80 relative percent survival in experimental challenge trials. Good protection was also demonstrated in long-term field trials where Atlantic salmon were naturally exposed to R. salmoninarum challenge until 23 months after vaccination. The same vaccine, which is licensed in Canada against BKD has also proved effective in reducing mortality from experimental challenge of coho salmon (Oncorhynchus kisutch) with Piscirickettsia salmonis, the causative agent of piscirickettsiosis. Under field conditions in Chile, use of the vaccine led to a significant reduction in piscirickettsiosis mortality in coho salmon over 10 months following sea transfer. The vaccine strain is unique in that it is the first live organism to be licensed as a vaccine for use in aquaculture. Potential mechanisms of protection against the two taxonomically disparate pathogens are discussed.

Identification and characterization of segments 3 and 4 of the ISAV genome.

Infectious Salmon Anaemia is a serious disease of farmed Atlantic Salmon on three continents. The disease causes severe anaemia and haemorrphagic liver necrosis, and carries major economic consequences for affected areas. Nevertheless, the causative agent, a novel orthomyxo-like Virus (Infectious Salmon Anaemia Virus - ISAV), is only partially characterized at the molecular level. We report the isolation and characterization of two novel ISAV segments at the genomic and proteomic levels. These segments are the third and fourth largest of the (ISAV) genome and may code for a nucleocapsid protein (NP) and a polymerase (PA). Western blot analysis using an ISAV polyclonal antibody identified one of these novel proteins as being the major tissue antigen. We discuss the implications of our findings for vaccine development and surveillance of Infectious Salmon Anaemia.

Characterization of attenuated Renibacterium salmoninarum strains and their use as live vaccines.

Two nutritionally mutant strains of Renibacterium salmoninarum (Rs) were isolated that grew on tryticase soy agar (Rs TSA1) or brain heart infusion agar (Rs BHI1). These 2 strains could be continuously cultured on these media, whereas typical R. salmoninarum would only grow on KDM-2 agar. We determined no other phenotypic difference that could be used to distinguish them from wild-type R. salmoninarum. Both strains were found to be avirulent when 5 x 10(6) bacteria were intraperitoneally (i.p.) injected into Atlantic salmon. Rs TSA1, Rs BHI1, and Rs MT-239 (a R. salmoninarum strain previously shown to be attenuated) were tested as live vaccines in 2 separate trials. The best protection was seen with Rs TSA1. Vaccinated Atlantic salmon had relative percent survival (RPS) of 50 at 74 d post-challenge in Trial 1 and 76 at 60 d post-challenge in Trial 2. In both trials, 100% of the control salmon died from bacterial kidney disease (BKD) (within 40 d for Trial 1 and 50 d for Trial 2) after i.p. challenge with 5 x 10(6) live cells of the virulent isolate Rs Margaree.

First identification of infectious salmon anaemia virus in North America with haemorrhagic kidney syndrome.

Haemorrhagic kidney syndrome (HKS), a serious disease affecting Atlantic salmon on the east coast of Canada, was determined to be caused by infectious salmon anaemia virus (ISAV) through the isolation of the pathogen on the SHK-1 (salmon head kidney) cell line and confirmation by ISAV-specific immunofluorescent antibody test (IFAT) and reverse transcriptase polymerase chain reaction (RT-PCR). In addition, the defining histopathology of HKS could be reproduced following the injection of material that rendered challenged fish ISAV-positive by cell culture in the absence of any other detectable pathogen. Preliminary nucleotide sequence comparison does not suggest any direct epidemiological connection between the Canadian and Norwegian isolates.

Absence of vertical transmission of infectious salmon anemia virus (ISAV) from individually infected Atlantic salmon Salmo salar.

Atlantic salmon Salmo salar L. eggs were collected from grilse that were individually identified as ISAV-positive based on the detection of pathogen in ovarian fluid by RT-PCR. The eggs were fertilised, disinfected and reared under quarantine conditions. To address the possibility of vertical transmission, fertilised eggs, alevins and parr were screened for the virus by SHK-1 cell culture and RT-PCR. In addition, ISAV-negative parr were injected with homogenates of potentially infected eyed eggs. ISAV was not detected in eyed eggs, alevins or parr. No mortalities occurred among fish injected with the egg homogenates. These observations suggest the absence of a vertical transmission route for ISAV infection.

PCR and probe-PCR assays to monitor broodstock Atlantic salmon (Salmo salar L.) ovarian fluid and kidney tissue for presence of DNA of the fish pathogen Renibacterium salmoninarum.

A simple, rapid PCR assay for the identification of Renibacterium salmoninarum in Atlantic salmon (Salmo salar L.) tissues detected DNA extracted from between 4 and 40 bacterial cells. PCR was at least as sensitive as culture when it was used to identify subclinically infected fish experimentally challenged with R. salmoninarum. However, PCR identified much higher numbers of kidney tissue and ovarian fluid samples from commercially reared broodstock fish to be positive for R. salmoninarum than did culture. This difference may be due to the antibiotic chemotherapy of broodstock fish used by the industry in 1994 to control the vertical transmission of R. salmoninarum. A much closer relationship between PCR and culture results was observed for ovarian fluid samples collected from broodstock fish in 1993. Also, PCR scored a much higher percentage of kidney tissue samples than ovarian fluid samples from 1994 broodstock fish positive for R. salmoninarum, which may reflect the uneven distribution of the pathogen in different fish tissues. Inclusion of a nested probe to identify the PCR-positive 1994 ovarian fluid samples increased the sensitivity of detection to between one and four cells and the number of samples that scored positive by almost threefold. These data indicate that many infected ovarian fluid samples contained very low numbers of R. salmoninarum cells and, because almost all these samples were culture negative, that PCR may have detected dead or otherwise unculturable bacterial cells.

Further characterization of Renibacterium salmoninarum extracellular products.

Renibacterium salmoninarum, the agent of bacterial kidney disease in salmonids, releases high concentrations of extracellular protein in tissues of infected fish. The extracellular protein consists almost entirely of a 57-kDa protein and derivatives of degradation and aggregation of the same molecule. The 57-kDa protein and its derivatives were fractionated into defined ranges of molecular mass. Separated fractions continued to produce degradation and aggregation products. One-dimensional electrophoretic separation of extracellular protein revealed a number of proteolytically active bands from > 100 to approximately 18 kDa associated with various 57-kDa protein derivatives in the different molecular mass fractions. Two-dimensional separation of extracellular protein showed that continued degradation and aggregation, similar both in location and behavior to some of the 57-kDa protein derivatives, was also displayed by the proteolytically active bands after their separation. Effects of reducing agents and sulfhydryl group proteinase inhibitors indicated a common mechanism for the proteolytically active polypeptides characteristic of a thiol proteinase. The results suggested that the 57-kDa protein and some of its derivatives undergo autolytic cleavage, releasing a proteolytically active polypeptide(s) of at least 18 kDa. Soluble polysaccharide-like material also was detected in extracellular products and tissue from infected fish. Antiserum to the polysaccharide-like material cross-reacted with O-polysaccharide of the fish pathogen Aeromonas salmonicida, suggesting some structural similarity between these polysaccharides. The polysaccharide and the proteolytic activity associated with the 57-kDa protein derivatives should be investigated with respect to the pathogenesis of R. salmoninarum infections.

Characterization of Aeromonas salmonicida variants with altered cell surfaces and their use in studying surface protein assembly.

Aeromonas salmonicida variants were characterized for alterations in their cell surface structure and used to examine reconstitution of the surface protein layer (A-layer). Variants lacking outer membrane O-polysaccharide were devoid of A-layer and excreted stainable floret-like material of the surface protein (A-protein). One variant, showing partial loss of O-polysaccharide, was associated with a disrupted A-layer and excretion of some A-protein. Variants lacking A-protein but possessing O-polysaccharide rapidly absorbed and concentrated sufficient excreted A-protein at the cell surface to coat the cells with a single confluent layer. Although differences in electrophoretic mobilities of A-proteins and O-polysaccharides from "typical" and "atypical" strains were evident, the different A-proteins and A-protein-deficient variants were interchangeable for reconstitution of a surface protein layer. No association of A-protein with cell surfaces of unrelated gram-negative bacteria was observed.

Characterization of Aeromonas salmonicida mutants with low-level resistance to multiple antibiotics.

Aeromonas salmonicida mutants selected for low-level resistance to small-molecular-mass antibiotics occur at frequencies that suggest point mutations and exhibit pleiotropic effects such as a multiple low-level antibiotic resistance, changes in outer membrane protein profiles, and loss of major exoprotease activity. Multiple low-level resistance appeared as the result of decreased outer membrane permeability associated with a change from a 38.5- to a 37-kilodalton (kDa) outer membrane protein. This decreased outer membrane permeability was determined by rates of nitrocefin hydrolysis by periplasmic beta-lactamase activity. The findings described above were supported by isolation of revertant strains selected for regained exoprotease activity, which also lost multiple low-level resistance and possessed outer membrane protein profiles indistinguishable from those of the original parent strains. Exoprotease from parent and revertant strains was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a major extracellular protein of approximately 69 kDa. No accumulation of a protein in this molecular mass range was observed in extracellular or periplasmic fractions from the mutants. The results suggested that exoprotease loss is not simply the result of an inability to export protease from the periplasm because of outer membrane protein changes, as has been reported for certain mutants of some other gram-negative bacteria. Also, several growth conditions were used, including some that have been reported to influence outer membrane protein expression and permeability in other enteric gram-negative bacteria. Although exoprotease expression in A. salmonicida was influenced by these conditions, no major outer membrane protein changes which would correspond to changes observed in the mutants were observed in parent strains.

Measurement of thyroid hormone in experimental thyroid tumours in rats.

Rats treated with 131I and propylthiouracil were shown to develop thyroid tumours 7--9 months after treatment. In this group, the levels of total thyroxine and tri-iodothyronine, and free thyroxine and tri-iodothyronine in the serum were low, and that of TSH was raised. In a group of rats treated with 131I and then propylthiouracil and thyroxine, thyroid tumours were found despite normal concentrations of total and free thyroxine and tri-iodothyronine in the serum. The level of TSH in the serum was significantly raised in this group. Thyroid tumours were not found in the various control groups of rats.