Rozprawy doktorskie na temat „Fish Pathology”

Aquaculture is a recent and fast growing food production industry. Development of this sector is essential for increasing export earning and improvement of Socio-Economic status of rural people. The fish and shrimp production by capture have not increased per unit effort over a decade. The excessive pressure on the capture sector by the increasing world population forced man to search for another alternative namely aquaculture, which presently contributes substantially to global fish production.

Legionella pneumophila was first recognized as a cause of human pneumonia in 1976 . Since then, much has been learned about the microbiology, pathophysiology and epidemiology of this organism. The features which permit one strain but not another to invade human lung tissue and produce disease remain incompletely understood. This study e valuated several attributes of a virulent and an avirulent strain of L. pneumophila in an attempt to identify characteristics which would distinguish the two. Evaluation of a new medium, buffered egg yolk agar, showed that virulence was maintained after 26 passages, which was the same as the buffered charcoal yeast extract agar used for comparison. However, growth appeared earlier and was heavier on the charcoalcontaining medium. Morphologically, the avirulent strain produced greater numbers of filamentous forms and was found to be encapsulated more frequently. Treatment with polymixin B produced morphologic changes similar to those previously reported but failed to alter the virulence of either strain. No plasmids were found in either strain nor were consistent differences in protein content demonstrated using sodium dodecylsulfate polyacrylamide gel electrophoresis. Both strains reacted less intensely as cultures aged with a monoclonal but not a polyclonal antibody in a direct fluorescent antibody assay . This change was more pronounced when the virulent organism was tested. Chemotactic assays showed similar tendencies when human or guinea pig mononuclear cells were compared to two estuarine species (hogchoker and spot) and one freshwater species (golden shiner minnow) of fish. In vivo results were also similar when two of the three species of fish were tested, suggesting that either the spot or the minnow may be used in evaluation of certain characteristics of L. pneumophila. Organisms were isolated from apparently healthy fish up to 15 days after inoculation in some instances. This suggests that fish may be a possible additional environmental reservoir for Legionella pneumophila.

Le cancer de l'endomètre est le cancer gynécologique le plus fréquemment diagnostiqué chez les femmes en Europe, aux USA et au Japon. Les mécanismes de carcinogenèse endométriale sont encore mal appréhendés et les facteurs histopronostiques classiques ne permettent pas toujours de prédire le risque évolutif. Dans ce travail, l'étude de l'expression des métalloprotéases (MMP-2,-7 et -9) et des inhibiteurs tissulaires des métalloprotéases (TIMP-1 et -2) suggère l'implication de ce système enzymatique dans le processus de carcinogenèse endométriale avec un profil d'expression différent dans l'endomètre normal, l'hyperplasie et le cancer de l'endomètre. Nous avons par ailleurs pu montrer que l'analyse de l'expression de MMP-2 (gélatinase A) et de TIMP-2 permettait de définir un sous-groupe de cancer de l'endomètre qui était particulièrement à risque d'extension locale et à distance. Nos résultats ont également permis de confirmer le rôle prépondérant de la perte d'hétérozygotie (LOH) de TP53 dans la genèse des cancers de l'endomètre de type II et la survenue plus tardive de cet évènement dans les cancers de l'endomètre de type I (hormonodépendants). En terme de pronostic, l'analyse du gène TP53 par FISH (hybridation par fluorescence in situ) n'est pas supérieure à celle obtenue par IHC (immunohistochimie). Enfin, l'étude des corrélations histopronostiques réalisée dans ce travail suggère que l'évaluation conjointe de la ploïdie, de MMP-2 et TIMP-2, de p53, du Ki67 et des récepteurs hormonaux (E et P) sur les prélèvements tissulaires pourrait mieux définir une population à haut risque évolutif pouvant bénéficier de thérapeutique ciblée.

Streptococcus agalactiae is a pathogen of multiple hosts. The bacterium, an aetiological agent of septicaemia and meningo-encephalitis in freshwater and saltwater fish species, is considered a major threat to the aquaculture industry, particularly for tilapia. Cattle and humans are however the main known reservoirs for S. agalactiae. In humans, the bacterium (commonly referred to as Group B Streptococcus or GBS) is a member of the commensal microflora of the intestinal and genito-urinary tracts, but it is also a major cause of neonatal invasive disease and an emerging pathogen in adults. In cattle, S. agalactiae is a well-recognized causative agent of mastitis. Numerous studies focusing on S. agalactiae from human and bovine origins have provided insight into the population structure of the bacterium, as well as the genome content and pathogenic mechanisms through identification of virulence determinants. Concerning S. agalactiae from aquatic origins, scientific information mainly focused on case reporting and/or experimental challenges, with a limited or absence of information in terms of pathogenesis, virulence determinants and genotypes of the strains involved. The objective of this study was to enhance our understanding of the molecular epidemiology, host-adaptation and pathogenicity of S. agalactiae in aquatic species, with particular emphasis on tilapia. Firstly, a collection of 33 piscine, amphibian and sea mammal isolates originating from several countries and continents was assembled, with the aim of exploring the population structure and potential host specificity of aquatic S. agalactiae. Isolates were characterised using pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and a standardised 3-set genotyping system comprising molecular serotypes, surface protein gene profiles and mobile genetic element profiles. Two major subpopulations were identified in fish. The first subpopulation consisted of non-haemolytic isolates that belonged to sequence type (ST) 260 or 261, which are STs that have been reported only from teleosts. These isolates exhibited a low level of genetic diversity by PFGE and clustered with other STs that have been reported only in fish. Another common feature was the absence of all surface protein genes or mobile genetic elements targeted as part of the 3-set genotyping and that are usually found in human or bovine isolates. The second subpopulation consisted of β-haemolytic isolates recovered from fish, frogs and sea mammals, and that exhibited medium to high genetic diversity by PFGE. STs identified among these isolates have previously been identified from strains associated with asymptomatic carriage and invasive disease in humans. The human pathogenic strain ST7 serotype Ia was detected in fish from Asia. Moreover, ST283 serotype III-4 and its novel single locus variant ST491 detected in fish from Southeast Asia shared a 3-set genotype identical to that of an emerging ST283 clone associated with invasive disease of adult humans in Asia. These observations suggested that some strains of aquatic S. agalactiae may present a zoonotic or anthroponotic hazard. STs found among the seal isolates (ST23) have also been reported from humans and numerous other host species, but never from teleosts. This work provided an excellent basis for exploration of the virulence of selected strains in experimental challenges. The virulence of two strains of S. agalactiae was experimentally investigated by intra-peritoneal infection of Nile tilapia (Oreochromis niloticus), using an isolate originally recovered from fish and belonging to ST260, and an isolate originating from a grey seal and belonging to ST23. The clinical signs, the in vivo distribution of viable bacteria and bacterial antigens, and the gross and histopathological lesions that developed during the time course of the infection were investigated. The ST260 strain was highly virulent, whereas no major clinical sign or mortalities occurred in the fish challenged with the ST23 strain. After injection, both strains however gained access to the bloodstream and viable bacteria were recovered from all organs under investigation. During the early stages of infection, bacteria were mostly found within the reticulo-endothelial system of the spleen and kidney. Thereafter, the ST260 demonstrated a particular tropism for the brain and the heart, but granulomatous inflammation and associated necrotic lesions were observed in all organs. ST23 was responsible for a mixed inflammatory response associated with the presence of bacteria in the choroid rete and in the pancreatic tissue only. After 7 days post-challenge and for both strain, the formation or containment of bacteria within granulomata or other encapsulated structures appeared to be a major component of the fish response. However, the load of viable bacteria remained high within organs of fish infected with ST260, suggesting that, unlike ST23, this strain is able to survive within macrophages and/or to evade the immune system of the fish. This work demonstrates that the lack of report of ST23 strains in fish is possibly not due to a lack of exposure but to a lack of virulence in this host. The two strains, which differ in prevalence and virulence in fish, provide an excellent basis to investigate genomic differences underlying the host-association of distinct S. agalactiae subpopulations. The genome of the ST260 strain used in challenge studies was sequenced. We therefore provided the first description for the genome sequence of a non-haemolytic S. agalactiae isolated from tilapia (strain STIR-CD-17) and that belongs by multi-locus sequence typing (MLST) to clonal complex (CC) 552, which corresponds to a presumptive fish-adapted subgroup of S. agalactiae. The genome was compared to 13 S. agalactiae genomes of human (n=7), bovine (n=2), fish (n=3) and unknown (n=1) origins. Phylogenetic analysis based on the core genome identified isolates of CC552 as the most diverged of all S. agalactiae studied. Conversely, genomes from β-haemolytic isolates of CC7 recovered from fish were found to cluster with human isolates of CC7, further supporting the possibility that some strains may represent a zoonotic or anthroponotic hazard. Comparative analysis of the accessory genome enabled the identification of a cluster of genes uniquely shared between CC7 and CC552, which encode proteins that may provide enhanced fitness in specific niches. Other genes identified were specific to STIR-CD-17 or to CC552 based on genomic comparisons; however the extension of this analysis through the PCR screening of a larger population of S. agalactiae suggested that some of these genes may occasionally be present in isolates belonging to CC7. Some of these genes, occurring in clusters, exhibited typical signatures of mobile genetic elements, suggesting their acquisition through horizontal gene transfer. It is not possible to date to determine whether these genes were acquired through intraspecies transfer or through interspecies transfer from the aquatic environment. Finally, general features of STIR-CD-17 highlighted a distinctive genome characterised by an absence of well conserved insertion sequences, an abundance of pseudogenes, a smaller genomic size than normally observed among human or bovine S. agalactiae, and an apparent loss of metabolic functions considered conserved within the bacterial species, indicating that the fish-adapted subgroup of isolates (CC552) has undergone niche restriction. Finally, genes encoding recognised virulence factors in human S. agalactiae were selected and their presence and structural conservation was evaluated within the genome of STIR-CD-17.

The cerebellum contains most of the central nervous system neurons and it is classically known to be a key region for sensorimotor coordination and learning. However, its role in higher cognitive functions has been increasingly recognised, thus raising the interest of neuroscience and neuroimaging communities. Despite this, knowledge of cerebellar structure and function is still incomplete and the interpretation of experimental results is often problematic. For these and also technical reasons the cerebellum is still frequently disregarded in magnetic resonance imaging (MRI) studies. Therefore, the principal aim of this work was to use MRI to investigate cerebellar microstructure and macrostructural connectivity in health and pathology, focusing also on technical aspects of image acquisition. The starting point of each project described in the present thesis were techniques, models and pipelines currently accepted in clinical practice. The meeting of inadequacies or problems of such techniques raised questions that pushed research to a more fundamental level. This thesis has three main contributions. The first part presents a clinical study of cerebellar involvement in processing speed deficits in multiple sclerosis, where combined tractography and network science highlighted the importance of the cerebellum in patients’ cognitive performance. Then a deeper investigation conducted on high-quality diffusion MRI data with advanced diffusion signal models showed that subregions of the cerebellar cortex are characterised by different microstructural features: this represents one of the very first attempts to use diffusion MRI to face the widespread idea of cerebellar cortex uniformity, which has been recently challenged by findings from other research fields, thus providing new perspectives for the study of cerebellar information processing in health and pathology. Finally, the emerging technical problems that hamper the study of small structures within the cerebellum were tackled by developing dedicated acquisition protocols that exploit reduced field-of-view techniques for 3T and 7T MRI scanners.

Les tumeurs solides sont souvent confrontées à un environnement déficient en oxygène, dit hypoxique. Hypoxia-Inducible Factor 1 (HIF1) est le facteur de transcription clé de l'adaptation cellulaire à l'hypoxie, régulant de nombreux gènes impliqués dans l'angiogenèse, le métabolisme cellulaire ou la régulation du pH. Ma thèse s'articule en trois axes autour de HIF1 et de la reprogrammation métabolique hypoxique. J'ai d'abord étudié Factor-Inhibiting HIF1 (FIH), l'un des deux senseurs d'oxygène régulant HIF1. Nous avons montré que FIH est essentiel dans le développement tumoral en inhibant à la fois l'activité transcriptionnelle de HIF1 et la voie p53-p21. J'ai ensuite étudié le " shift " du métabolisme cellulaire vers la glycolyse induit par HIF1, générant une addiction pour le glucose. Nos travaux ont montré que paradoxalement, les cellules hypoxiques synthétisent du glycogène via HIF1 constituant ainsi une réserve de glucose intracellulaire. Le glycogène confère alors une résistance accrue des cellules tumorales suite à une carence en glucose. Enfin, j'ai pu montrer que l'AMPK, " gardien de la balance énergétique ", n'est pas nécessaire au maintien d'un niveau viable d'ATP suite à l'inhibition de la glycolyse, via le blocage de l'export de lactate, mais exerce, un effet protecteur en absence de glucose. Cependant, l'inhibition conjointe du transporteur de lactate, MCT4, et de l'AMPK réduit fortement le développement tumoral dans un modèle de xénogreffes chez la souris, suggérant un rôle crucial de ces deux acteurs dans ce contexte. L'ensemble de ces travaux a permis d'identifier plusieurs cibles potentielles impliquées dans la plasticité métabolique en hypoxie.

Food and water are the two major routes through which aquatic organisms are exposed to exogenous hazardous toxic insults. The pathology of the toxic impacts due to a food contaminant (aflatoxin B, or AFB,) and an environmental pollutant (cadmium) was studied in pearl spot, Etrop/us suratensis (Bloch). The influence of these two toxicants on various physiological indices as well as vital organs of the fish was studied in two separate experiments by exposing the fishes to either 400 ppb of AFB, or 9.4 ppm of cadmium for a period of 8 weeks. The physiological alterations due to aflatoxin exposure includes, an initial decrease followed by an increase in erythrocyte count; increase in leucocyte count, packed cell volume (PCV), serum alkaline phosphatase, aspartate aminotransaminase (AST) and alanine aminotransaminase (AL T); and a decrease in serum proteins. Histopathological and ultrastructural studies revealed progressive degenerative changes in the vital organs, liver, kidney, spleen and thymus. Liver sections revealed biliary proliferation and presence of pleomorphic, polyhedral and basophilic megalocytes invading into the normal parenchyma, which was diagnosed as hepatocellular carcinoma. The electronmicrographs revealed dilatation, fragmentation , proliferation and whirl formation of endoplasmic reticulum (ER), mitochondrial damages like condensation and loss of cristae and granules, nuclear changes like presence of perichromatin and chromatin granules, electron dense inclusions and presence of autophagosomes in the cytoplasm. The toxic insults due to cadmium were manifested as increase in ESR, AST and AL T and a decrease in PCV, serum proteins and alkaline phosphatase activity. Liver, kidney, spleen, thymus and gills exhibited severe degenerative changes at cellular and subcellular levels. Liver revealed coagulative necrosis and fibroblastic proliferation . The renal tubular epithelial cells were desquamated and periglomerular thickening as well as increased nuclearity were observed in glomeruli. The secondary lamellae of gills were elongated and fused together in cadmium exposed fishes. The major ultrastructural changes in the vital organs include ER fragmentation, mitochondrial swelling, presence of multivesicular bodies, autophagia, damage to desmosomes and cell membranes.

Aquaculture has become one of the expanding food production method in the world. Its growth rate in the last decade has been phenomenal recording 9.4% per year from 1986 to 1995 (FAO 1977). Global aquaculture production totalled 27 .8 mmt and aquaculture production of fi sh and shellfish reached 20.9 mmt in 1995. India is a major fish producing country in the world. Aquaculture production of fish and shellfish in India increased from 686260 metric tonnes to 1608938 metric tonnes over the period 1986 to 1995. In aquaculture, India holds the second position in the world. Aquaculture contributes substantially to world food used for direct human consumption. Percapita food fish supply from aquaculture has increased by 163% since 1984, from l.4kg to 3.68kg in 1995. With the marine fish production reaching a plateau, any further demand for fish has to be met from aquaculture only. In order to reduce the gap between supply and demand of food fish development of ecofriendly and sustainable aquaculture is very important. Fish and crustaceans are generally safe food for human. However, the risk of contamination of fishery products by microorganisms, chemicals etc. is greater in farming systems compared to open seas. Food safety hazards due to microorganisms are significant from human health consideration. Bacteriological hazards include pathogenic bacteria associated with food-borne disease. There are two broad groups of pathogenic bac teria of public health significance that will contaminate products from aquaculture - those th at are indigenous to the aquatic environment from which fi sh/shellfish are harvested and those introduced into fish/shellfish as a result of environmental contamination resulting from di sposal of sewage and land run-off. In addition to this, pathogens are also introduced into fish and fishery products during post harvest handling and processing. One major pathogen associated with food poisoning is Clostridium botulinum .

Studies on the pathobiology of penaeid larvae and postlarvae. One of the major fac~ltors which influences the production and quality or seed in the hatchery and their subsequent culture in the grow-out system is tl!e uisczses and parasites affecting the stock. Under certain un fa-~surnble c~nditions~ diseases due to biotic and abiotic factors affect the iavYse ana postlarvae of penacid prawns. Further, these developing stages ale founu to be rrisre susceptible to diseases than the adult.

Oil and gas exploration in Northern Canada uses explosive-based seismic techniques to locate hydrocarbon reserves beneath waterbodies not frozen to the bottom. The use of explosives in, or near, waterbodies has the potential to harm fishes, primarily through instantaneous pressure changes (IPCs) generated from the detonations. These IPCs can damage soft tissues through the rapid compression and expansion of the swimbladder as the pressure wave passes. In Canada, a document entitled Guidelines for the Use of Explosives In or Near Canadian Fisheries Waters recommends that peak pressures not exceed 100 kPa for the protection of fish, however damage has been reported below this level. To simulate seismic exploration and examine potential pathological changes surrounding the current Guideline, fish across different developmental stages and with varying degrees of swimbladder presence were exposed to a variety of explosive based IPC levels in field experiments. Early life stages of rainbow trout (Oncorhynchus mykiss) including eyed eggs, sac fry, and juveniles were caged and exposed to discrete detonations from 0 to 280 kPa in the Mackenzie Delta, NWT. These fish were subsequently examined for both gross pathological and histological changes to cranial structures as well as swimbladder, kidney, liver and gill tissue. Results showed changes in both the area and circumference of the cranial region of eyed eggs, as well as swimbladder, ocular and kidney damage in juveniles. Additionally, caged adult swimbladder bearing lake trout (Salvelinus namaycush) and non-swimbladder bearing slimy sculpin (Cottus cognatus) were exposed to explosive based IPCs ranging in peak pressure from 0 to 127 kPa at the Experimental Lakes Area, Ontario. Fish were later examined grossly and blood, liver, kidney, intestine, and spleen were examined to determine the presence of any traumatic based pathological changes. Results indicated the occurrence of swimbladder hemorrhage in lake trout exposed to IPCs near the current Guideline level. Finally, a risk assessment for lake trout of the Mackenzie Delta exposed to IPCs was undertaken; to examine the potential for adverse risk to individuals and populations, and the likelihood of populations being unable to recover. Based on the findings of the aforementioned studies, the recommended Guideline level is not protective of early life stages of rainbow trout and furthermore represents the threshold at which damage to the swimbladder in adult lake trout does not occur, as such, a re-examination of the recommended Guideline level is warranted.

Edible oyster populations of the Indian coast are currently naive and are not subjected to live introductions or trans-boundary movements. There were no previous reports on edible oyster mortality due to disease out breaks in the country. Still, the major reported parasites in oysters over the world have been observed in the present study within a low prevalence. The two OIE listed pathogens, Bonamia ostrea and Marteilia refringens, the protozoan, Perkinsus beihaiensis, the trematode, Bucephalus sp. and the shell parasite Polydora sp. are those that have the potential to be a problem for the future farming. Hence a comprehensive screening for pathogens in other beds throughout the coastline is strongly recommended. Presently oyster farming is getting popularized and is being extended to more and more regions along the coast. At this stage, a good baseline data on parasites and pathogens within natural environments is necessary for proper disease management. The baseline data generated in this study will be helpful for planning a health monitoring program in Crassostrea madrasensis The farming site at Sathar Island was found to have the advantage of producing oysters with high condition index and low incidence of parasites. The duration of farming limiting to one year was found to be ideal since it reduces the exposure period for parasite entry. But the presence of the major protozoan parasites in the ecosystem should be considered while expanding and intensifying oyster culture in future as the stressful condition can cause parasitic proliferation in the ecosystem. A similar situation was observed in mussel farming grounds along the west coast of India where a proliferation of OIE listed protozoan, Perkinsus olseni was associated with intensification of mussel culture. The nested primers developed for the major protozoan parasites in this study can be used for screening the stocks for selecting brood stock and for planning farming sites. As PCR screening using these primers is more sensitive than the conventional methods, movement of infected stocks to uninfected regions can be minimised based on this screening In conclusion, Crassostrea madrasensis stocks along the south Indian coast (with respect to the stations studied) were found to be safe with a low prevalence of pathogens. But, the comparatively higher burden of parasites and pathological conditions observed in Tuticorin Bay indicates the impact of contaminant exposure. Thus for maintaining the good health status of oyster stocks, periodic surveillance and monitoring are necessary and also the water quality should be regularly monitored for better control over the chances of disease out breaks especially around the cultured areas.

Ph.D.
Members of Cichlidogyrus are monogenean ectoparasites occurring on the gill filaments of mainly cichlid fishes. An overview of the taxonomic background of the genus and motivation for the study is provided. In this study, existing information on their distribution is organised in a comprehensive table which includes 85 species, their taxonomic authors, hosts and localities from which they have been recorded. Representatives of this genus occur mainly in Africa, but have been found on cichlids as far as Mexico. Host specificity of members of the group is discussed. During the present investigation specimens of the genus Cichlidogyrus were found parasitising Pseudocrenilabrus philander philander, in the Padda Dam in Johannesburg, Gauteng, South Africa. Specimens were examined and identified as Cichlidogyrus philander, previously described from the same host, from Lake Kariba, in Zimbabwe. The morphology was studied utilizing light – and scanning electron microscopy. Specimens were stained with Horen‟s trichrome and photographed. Furthermore 100 whole mounted specimens, which were fixed and mounted in glycerine-ammonium-picrate, were measured. Larval specimens, and mature individuals with eggs in situ, were examined. The investigation of the ecology entailed monthly surveys during which a total of 245 fish specimens were collected with the aid of hand nets, over a period of 14 months (from March 2006 to June 2007). The fish were transported to the laboratory and killed by severing the spinal cord. Gills of specimens of P. p. philander (111), Gambusia affinis (105), Tilapia sparmanii (28) and one (1) 2 specimen of Cyprinus carpio were inspected, for the presence of monogeneans, with the aid of a dissection microscope. Only members of P. p. philander were parasitised by C. philander, indicating strict host specificity. The number of specimens varied from 0 to 184 per fish. The highest mean intensity of 91.6 occurred in the winter month of June 2007, although no significant correlation between season and the occurrence of this parasite was recorded. The prevalence was 100 % for ten of the fifteen surveys and no preference for host sex was recorded. The intensity of the infection correlates positively to the total length of the host, i.e. larger hosts harbour more worms. A positive correlation between condition factor and the total number of worms was found, indicating that more parasites are found on fish with a normal condition factor. No significant difference occurred between the numbers of parasites collected from gill arches on the left or right sides of fish. Statistical analysis of data showed that parasites disperse across the four gill arches with a higher proportion of parasites on the third and second gill arch. The highest proportion of the collected parasites occurred on the dorsal regions and on the distal ends of the gill filaments. Water quality variables do not significantly correlate to the prevalence, abundance or mean intensity of this parasite. This is the first ecological study conducted on specimens of C. philander, worldwide.