The following report is a summary of an investigation undertaken on behalf of the Canadian fishing industry, more especially the inland fisheries of the Prairie provinces, by the Fisheries Research Board of Canada. The investigation was allotted to the Pacific Fisheries Experimental Station at Vancouver, the actual work being performed in the Physics Department of the University of British Columbia. The application of physical methods to biological and other problems is becoming more prevalent. The case in hand is an example of the application of physics, more particularly the laws of light scattering, to determining the infestation of fish by Triaenophorus crassus. The arguments wherever possible have been stated in everyday language and notwithstanding the possibility of boring the informed reader, all experiments are described in detail. This, it is hoped, will accomplish two purposes. Firstly the biologist will have a better understanding of the investigation and secondly the application of physics to similar problems can be assessed. Included at the end of the section on candling is an attempt to explain the formation of shadows by objects imbedded in turbid media. The argument is first given, in simple language followed by a mathematical discussion. The complexity of the theory of scattered light in terms of ultimate processes need not be stressed. A treatment based on the theory of M. Born is found in Appendix I. / Science, Faculty of / Physics and Astronomy, Department of / Graduate

Nine isolates of bacteria recovered from fish dying at marine facilities were collected from different geographic areas. The strains included: an isolate from chinook salmon (Oncorhynchus tshawytscha) reared in net pens in New Zealand, an isolate from chum salmon (Oncorhynchus keta) held at a laboratory in Oregon, USA., and seven strains recovered from tilapia (Oreochromis spilurus), silvery black porgy (Acanthopagrus cuvieri), and greasy grouper (Epinenhelus tauvina) cultured in Kuwait. All isolates were characterized by examination of morphological and biochemical properties and were confirmed to be members of the genus Vibrio. All isolates differed phenotypically from each other, from vibrios known to be pathogenic for fish, and from other named Vibrio species. Analysis of key phenotypic characteristics used to establish existing species suggested that the isolates tested were new Vibrio species. Four of the isolates (two from coldwater fish and two from warmwater fish) were selected for further study. This included determination of percent guanine plus cytosine (%G+C), comparison of growth characteristics, analysis of major 0 antigens and testing of pathogenicity. The four isolates examined had an absolute requirement for NaCl. Optimum growth temperatures varied among the isolates and were consistent with the temperature optima of the hosts from which the isolates were obtained. Serological analysis using slide agglutination, microtiter agglutination, and Ouchterlony double diffusion tests detected specific thermostable (0) antigens unique for each of the four isolates. A common minor antigen was observed between two of the other isolates from Kuwait. Experimental infections were produced in fingerling rainbow trout (Salmo gairdneri) using intraperitoneal injection of the four isolates. The pathogenicity of the two isolates from Kuwait was higher than that of the two salmonid isolates. The strains from Kuwait were used to challenge juvenile chinook salmon by waterborne exposure. The pathology produced by infection was characteristic Gram-negative hemorrhagic septicemia. / Graduation date: 1988

Isolates of Yersinia ruckeri were obtained from Europe, North America, Australia and South Africa. The biochemical and serological characteristics of the isolates were investigated. Biochemically the isolates were extremely uniform although motile, Tween positive isolates could be differentiated from non-motile, Tween negative isolates; these were designated biotypes 1 and 2 respectively. With the exception of two isolates, biotype 2 isolates were confined to the U. K. Five 0-serotypes were recognised and an O-serotyping scheme is proposed; the relation of this scheme to previously described schemes is discussed. The geographic distribution of the different serotypes is also discussed. The lipopolysaccharide (LPS) and outer membrane protein (OMP) profiles of isolates were analysed by SDS-PAGE and Western-blotting using both rabbit and rainbow trout antisera. The relation of LPS-type to 0- serotype, as well as variation within LPS-types, is discussed. Based on interstrain variation in the molecular weight of a heat-modifiable protein and of peptidoglycan-associated (porin) proteins, an OMP-typing scheme was developed. Three major OMP-types comprised 95% of the isolates studied. Variation in biotype, serotype and OMP-type was used as an epizootiological tool, and six serotype 01 clonal groups were recognised which differed in their geographic distribution. The production of iron-regulated OMPs and siderophores was investigated. Four iron-regulated OMPs were produced in all of the isolates examined; siderophores appeared not to be produced by any of the isolates. Production of iron-regulated OMPs was not an important virulence determinant and appears to be a chromosomally-mediated factor. Resistance to the bactericidal effects of normal rainbow trout serum and virulence were also investigated. Serum-resistance was associated principally with two serotype 01 clonal groups and virulence was associated with the same two clonal groups. Other serotype 01 clonal groups and other serotypes were iii generally serum-sensitive and avirulent. Thus, serum-resistance is an important virulence determinant in this organism. The role of outer membrane components in serum-resistance and virulence is discussed.

Samples of Gasterosteus aculeatus from 16 areas in the Fraser Valley and environs were examined for plerocercoids of Schistocephalus solidus. Fish at Coal Harbour and Alouette Lake were sampled several times over a twelve month period. The number and sizes of worms present in the Alouette Lake fish samples were recorded, and it was found that infected fish less than 45 mm. total length carried on average more worms than those over 45 mm., and that uninfected adult fish were caught only during the breeding season. In another lake, infected fish were found in a different area from the uninfected ones. The fish intermediate host was shown to be affected by the infection in four ways: (i) Infected fish died sooner than uninfected fish, (ii) Heavily infected fish were lighter in weight of fish tissue than controls of the same length, (iii) The total standard respiration rate of infected fish was higher than that calculated by combining values obtained from uninfected fish and published values for in vitro plerocercoids. (iv) Heavily infected fish required up to twice as much oxygen per gram fish weight per hour when swimming at the same speed as control fish. Other aspects were examined but the results were inconclusive or negative. The observations on natural populations are discussed in the light of the experimental findings. / Science, Faculty of / Zoology, Department of / Graduate

Graduation date: 1954

A number of aspects of the melano-macrophage centres of fish were investigated. In teleosts these centres consist of aggregates of pigment-laden macrophages and various leucocytes. They are usually embedded in haemopoietic tissue (mainly within the spleen and kidney) in association with the blood-supplying vessels and in a few species a distinct lymphoid cuff surrounds the entire structure. Salmonids are exceptional in that their melano-macrophages are scattered throughout the haemopoietic tissue and do not form distinct aggregates. At least four types of pigments have been described to occur in these phagocytes, viz. melanin, the lipogenic pigments ceroid and lipofuscin, and the haematogenous pigment haemosiderin. In light microscopy the centres appear in varying hues of yellow, brown and black. It has been suggested that these centres could well represent the primitive analogues of germinal centres of the lymph nodes of birds and mammals. As all previously available information came exclusively from a restricted number of teleost species it was considered of primary importance to carry out a study of the distribution and cytological organization of these centres in living representatives of Agnatha, Chondrichthyes and Osteichthyes. Seventy-two species of fish had their haemopoietic tissues examined by light microscopy for pigment-containing macrophages. Except for the lamprey Lampetra fluviatilis, all fish species were observed to possess these pigment cells. An evolutionary pattern was evident in both the distribution and the degree of organization of melano-macrophages; three major evolutionary trends were discernible, viz: i. a progressive increase in the abundance of pigment cells; ii. a structural evolution from a random distribution of individual pigmented macrophages observed in Agnatha and Chondrichthyes to organized centres characteristic of all Osteichthyes except the salmonids; iii. a change in organ location of these pigment cells from the liver in Agnatha, Chondrichthyes and the primitive bony fishes to the spleen and kidney in the advanced bony fishes. The increasing sophistication in cytological organization of the melano-macrophage centres is concomitant with the increasing levels of complexity of the cytoarchitecture of the lymphoid system. The increasing proclivity of the centres for the main lymphoid organs follows closely upon the evolution of the lymphoid system and represents a major advance in the evolution of lympho-reticular relationships. These analogies provide additional evidence that these centres are of a lymphatic nature and may well represent the primitive analogues of germinal centres of birds and mammals. Ontogenically pigment-bearing macrophages appear following upon first feeding. Immunological maturity appears to be attained at first feeding and the fact that it is shortly afterwards that melano-macrophages appear within the lymphoid tissues (eventually leading to melano-macrophage centre formation within them), seems to add weight to the evidence of a structural and functional relationship between melano-macrophage centres and lymphoid tissues. The very marked changes observed in the melano-macrophage centres during cachexia provided a convenient tool for studying these pigmented macrophages. In adult rainbow trout Salmo gairdneri and plaice Pleuronectes platessa kept at 12°C, the density of melano-macrophages and melano-macrophage centres respectively had increased considerably after 6 weeks of complete deprivation of food and by 10 weeks very high densities were observable. At higher temperatures (25°C), employing Tilapia zillii and swordtails Xiphophorus helleri, a very marked increase in the density of these centres was already evident after 3 weeks of complete starvation. With first feeding rainbow trout fry kept at 12°C, high densities of melano-macrophages were observed within the spleen and kidney after 3 weeks of complete starvation. No other treatment employed in this study was observed to induce any significant changes in the melano-macrophages of either fingerlings or adult fish. These results suggest that tissue atrophy is a major factor contributing to the formation of the pigments observed within the melano-macrophages. Electron microscopic observations employing normal and cachectic plaice indicated the following possible modes of origin for the pigments within melano-macrophages: i. melanin granules seem to be derived from their being simply phagocytosed from the classical melanin-containing cells that have been ruptured or otherwise damaged; ii. lipogenic pigments appear to derive from damaged cellular components such as effete mitochondria through the process of peroxidation of their unsaturated lipids; iii. haemosiderin is almost certainly derived from the breakdown of haemoglobin from effete erythrocytes. Since lipid peroxidation and recycling of iron compounds lead to the formation of free radicals and cations, these potentially toxic entities are bound to arise spontaneously within melano-macrophage centres. In view of this there is raised the possibility that the melanin within the centres could be playing a very important role through its well-recognized ability to absorb free radicals and its strong affinity for cations. While this would account for the presence of all these types of pigments within melano-macrophages, it could also explain why pigment cells are so often observed at sites of infection or tissue injury. All ingested cellular debris appears to be subjected to lysosomal enzyme activity and it is the indigestible residues (indigestible unsaturated lipids mainly) that give rise to the pigments which gradually accumulate. The absence of pigment in young larval fish, its steady accumulation with age in clinically normal fish and its presence without exception in older fish indicate that the pigments being studied seem to satisfy the criteria set forth for a basic biological aging process. The role of melano-macrophage centres in iron storage in normal and diseased fish was studied. The spleen, kidney and liver centres of fourteen species of clinically normal teleost fish were examined histochemically for haemosiderin. This was found to be present in varying amounts within the splenic centres of most specimens, but in contrast it was rarely found in the centres of the kidney and the liver. Linder conditions of starvation and in diseased fish, a markedly increased deposition of ferric iron occurred in the splenic centres of nearly all fish examined. By comparison, the iron content in the kidney and liver centres was generally still very low. These results suggest that although the centres in the various haemopoietic organs resemble each other morphologically and in their relation with the associated tissues, there could well be important functional differences between the centres of different organs. When rainbow trout that had been splenectomised were starved, accumulation of haemosiderin was diverted to the kidney melano- macrophages; the liver was still virtually devoid of iron. The possible implications of these findings are discussed. In conclusion, all the available evidence seem to indicate that the melano-macrophage centres of fish pnd the germinal centres of birds and mammals are similar in many ways. A major difference is the high levels of pigments in the centres of fish. This seems to be related to the inability of the latter to control their body temperature. Fatty acids of living organisms shift towards greater unsaturation under lower environmental temperatures as a means of maintaining protoplasmic viscosity within the range necessary for normal metabolic processes. Thus fish, because of their poikilothermic nature, have high levels of unsaturated fatty acids in their bodies and are thus more prone to lipofuscin formation. It has also been suggested that intracellular digestive processes of fish macrophages may not be well developed on the evolutionary scale. This could also lead to increased accumulation of indigestible materials within the melano-macrophage centres. Finally these results are discussed with special emphasis being placed on the following three points: i. that the melano-macrophage centres should be regarded as sites where a large variety of materials are aggregated, processed, sifted and disposed of in a variety of ways rather than regarding them as static areas passively accepting and storing any materials that come their way. Of special significance are those materials that are required for recycling such as iron-containing compounds; ii. that there are important functional differences between the melano-macrophage centres of different organs; iii. that as more information becomes available the melano-macrophage centres may well become useful as sensitive indicators of the state of health of the fish. A technique for bleaching the pigments within melano- macrophage centres in ultra-thin sections was developed. Treatment of the sections with permanganate for five minutes followed by fifteen minutes in metabisulphite resulted in complete bleaching of almost all the pigments.

No description available.

This study has been carried out to investigate the biological and environmental parameters influencing the settlement and post-settlement survival of the infective stages of Lepeophtheirus salmonis Kroyer 1837. The abiotic factors investigated were temperature and salinity. Temperature was found to have a significant effect on the settlement success of the copepodids with an inverse relationship between temperature and settlement. Survival of the louse at 10 days post infection showed a decrease at the reduced temperature. Temperature was also shown to have a direct relationship on lice development; higher seawater temperatures resulted in faster development. Regression analysis of temperature and settlement shows a significant correlation. A constant reduced salinity, 24%, resulted in a reduced ability of the copepodid to infect its host compared with 34%. Post-settlement survival in 24%, at approximately 13°C resulted in 5.8% survival of lice to day 10 post-infection compared to 79% in 34% salinity. When this experiment was repeated but with elevated seawater temperatures of up to 18°C, survival at the reduced salinity was found to be 75.3%, higher than the ambient control group. The developmental rate at day 10 post-infection of L.salmonis larva at 24% was shown to be slower than development at 34%o. Distribution of the L.salmonis copepodid on its host showed the highest settlement on the gills and on the fins, particularly the pectoral and dorsal fins. Examination of L.salmonis survival at day 10 post-infection indicated the highest losses on the gills and the pelvic, caudal and dorsal fins. Settlement on the pectoral fins showed the highest settlement and the greatest survival. The infective copepodid has a reduced ability to infect its host after 7 days following the moult from nauplius 2, compared to copepodids aged 1 and 3 days following the nauplius 2 moult. For copepodids of all ages, once settlement had been achieved, survival at 10 days post-infection was approximately 50% in all groups. Copepodids of all ages did not show any difference in the development rate at 10 days post infection. Highest settlement was found to be on the gills and pectoral and dorsal fins. The effects of varying dose rates of copepodids, has shown that a finite percentage of lice settle and survive the first five days post-infection. Settlement distribution was found to be highest on the body, gills and pectoral and dorsal fins. In serial infections of fish there was a reduced settlement count with second infections, possibly through intraspecific competition. Experiments using different host stocking densities showed that with an increased number of hosts the intensity of the infection of individual fish was reduced. Smaller fish appear more susceptible to settlement of L.salmonis than larger fish, and this is associated with the relatively greater fin area of those fish compared to larger fish. L.salmonis exhibits a preference for the fins as an area of settlement in all sizes of fish. Comparison of copepodid settlement on salmon and sea trout showed that in single populations of fish salmon had the highest intensities of infection whilst in mixed populations of fish sea trout had a higher intensity. Settlement distribution of L.salmonis on salmon showed greatest settlement on the body, pectoral and dorsal fins, whilst on sea trout settlement was highest on the body, pectoral, pelvic, caudal and dorsal fins. The comparative development of L.salmonis between the two species of host fish showed an increased rate of development on salmon. The calculated energy levels for L.salmonis larval stages show a decrease in available energy within each developmental stage. After 6 days from the nauplius 2 moult the copepodid starts to show a sharp decline in energy levels which coincides with the reduced ability of the copepodid to infect the host. Post-settlement energy levels remain constant even though the copepodid is actively feeding, as seen by SEM examination at 2 days postinfection. The principal lipid class found within L.salmonis larval stages as energy reserve is triacylgylcerol (37.6% of the total lipid). A preliminary epidemiological model for sea lice population dynamics is proposed. This is a differential equation compartmental model that has been designed to examine the flow of L.salmonis developmental stages on the host. The model was able to predict the timing of the maximum number of pre-adult 1 lice stages to within one day. The difference between the observed data and the model output is probably due to the considerable variability in the parameters used in the model construction.

Flavobacterium psychrophilum (Fp), the aetiological agent of rainbow trout fry syndrome (RTFS) and bacterial cold-water disease (BCWD), is responsible for significant mortalities and economic losses in the salmonid aquaculture industry worldwide. Currently, there is no effective commercial vaccine against RTFS available, and the treatment of the disease depends on the oral administration of a wide range of anti-microbial compounds, some of which have proven ineffective. With unsuccessful disinfection procedures, possibilities of antibiotic resistance developing and no commercial vaccine available, there is an increased need to rapidly detect Fp and reduce mortalities in the industry by improving control measures in the farm system. The aim of this thesis was to investigate possible sources of Fp in a rainbow trout fry farm system and to use this data to develop strategies to reduce the prevalence of the pathogen with this farming system. Novel assays to detect Fp (loop-mediated isothermal amplification; LAMP), quantify Fp (quantitative PCR; qPCR) and to detect the fishes’ host response to Fp (Luminex™) were developed, and then used alongside bacterial culture and nested PCR to determine the prevalence of Fp on a commercial fish farm. Four batches of eggs from 3 different geographic sources were collected on arrival at the farm and tested for the prevalence of Fp. Fry from these batches were monitored as they grew and were moved to different sites at the farm. Kidney, spleen and blood were collected at 3 different life stages from the fry, until they were sold for ongrowing by the farm. Water samples from the inlet, outlet and fry tanks were collected at each sampling point. PCR analysis and bacteriology were the two main methods selected for screening the eggs and fry tissue for Fp. All sources of eggs were found to be positive for Fp with prevalences ranging from 1.1 % - 1.9 % and there was a significant increase in prevalence over time for all 4 batches of eggs ranging from 19.8 % - 34.6 % by the final life stage sampled. There was also a substantial difference in the numbers of fry samples positive for Fp depending on whether nested PCR or bacterial culture were used, as well as the organ (kidney or spleen) tested. This highlighted the importance of sampling both organs rather than just the one. Nested PCR was more sensitive than culture with 13 % of the fry samples reported as Fp positive, by sampling both the kidney and spleen collectively, while only 5 % were Fp positive by bacteriology. The levels of Fp in all samples could not be quantified by qPCR due to limits in the sensitivity of the assay. For those samples that were quantified at the levels of Fp detected by qPCR ranged from 3.38 x 104 well-1 - 2.07 x 106 well-1 genome copies in egg samples; from 3.38 x 103 well-1 – 3.07 x 107 well-1 genome copies well-1 in tissue samples (spleen or kidney), and from 7.89 x 103 – 7.22 x 104 genome copies well-1 in water samples. The sensitivity of the standard curve was limited to 103 copies well-1 and following optimisation of the assay the annealing temperature was decreased by 1˚C to 62°C to reduce the cross-reactivity to negligible levels, though this reduced the sensitivity of the assay even further to 104 copies well-1. The detection limits by qPCR obtained by spiking samples with known amounts of Fp were 192 CFU mg-1 from egg samples, 184 CFU mg-1 from fry tissue samples, and 220 CFU ml-1 from water samples,. The sensitivity of the LAMP assay determined by spiking egg, kidney, spleen and water samples was 18 CFU mg-1, 22 CFU mg-1, 25 CFU mg-1 and 16 CFU ml-1, respectively. The latter was similar to, though not as sensitive as nested PCR. Nested PCR limits determined by spiking egg, kidney, spleen and water samples were 14 CFU mg-1, 11 CFU mg-1, 13 CFU mg-1 and 11 CFU ml-1. No cross-reactivity was found with any bacteria including other Flavobacterium species with nested PCR but cross-reactivity with other Flavobacterium species were found with both qPCR (1.51 % with Flavobacterium aquatile and 0.30 % with Flavobacterium johnsoniae) and LAMP. The LAMP assay showed slight cross-reactivity with Flavobacterium columnare and Flavobacterium branchiophilum. A novel Luminex™ assay was also developed and optimised, using microspheres coated with Fp, to detect antibodies to Fp in the serum of the fry. The Luminex™ allowed small volumes of serum from individual fry to be used to evaluate antibody response as an indirect method to determine exposure to and infection by Fp. A large number of fry from all 4 batches (88% - 94%) of eggs were found to contain anti-Fp antibodies though it still remains to be determined whether the antibodies were specific to Fp. From the work carried out in this study, it can be concluded that whether eggs are carrying Fp inside and/or on their surface, it should be possible to reduce the prevalence of Fp in farm systems by regularly screening the broodstock, eggs and fry. Supply of Fp-free eggs and milt is essential to reduce the reservoir of Fp on farms. Both the qPCR and LAMP assay require further optimisation but they do offer potential for the future screening of Fp at farm sites and in the laboratory. Future control measures for RFTS should include the screening of broodstock and eggs by sensitive methods so that Fp-free seed can be supplied to farms. This, alongside effective disinfection procedures, rigorous husbandry practices and future vaccine development will all be required to manage this very significant fish disease.

The epidemiology of Aeromonas salmonicida subsp. salmonicida in the marine environment was investigated. Nutrient resuscitation and infectivity studies did not support a previous claim of dormancy in A. salmonicida and validated the use of colony-forming units (cfu) in survival studies. Survival of A. salmonicida in seawater was assessed and found to be of short duration «10 days). Survival of the bacterium in non-sterile sediment, obtained from beneath a salmon cage, appeared to be limited. The minimum infective dose of A. salmonicida to Atlantic salmon in short duration (1-3 days) bath exposure in sea water was 10' cfu ml-I. Prolonged exposure for three weeks resulted in infection with 102 cfu ml- I. Intragastric intubation of the bacterium established infection with doses >105 cfu. Shedding of A. salmonicida from infected salmon was 105-108 cfu/fish/hr. Survival and shedding results were combined in a computer model. A. salmonicida was predicted to travel >6 km suspended within the water column of a sea loch. Covert infection in freshwater farmed salmon was assessed by ELISA and the standard stress test. Results indicated that ELISA may be useful as a routine monitor of furunculosis infection. The efficacy of dot-blot immunoassay was found to be 108 cfu A. salmonicida in fish kidney tissue. Rainbow trout (Oncorhynchus mykiss) and salmon mucus were not found to inhibit the growth of A. salmonicida supporting recent evidence that fish skin is a site of carriage. In vitro studies suggested that trout serum proteins do not confer protection from fish antibody on A. salmonicida in covert infections. Preliminary work was undertaken to develop a specific DNA probe for A. salmonicida which will allow its detection in environmental samples and carrier fish. A gene library of A. salmonicida was constructed in lambda gtll and screened for "A"-protein with antibodies.