Abstract
The present thesis concerns the study of myxobacteria infections from the bacterium Tenacibaculum maritimum in farmed marine fish in Greece and the accomplishment of experimental infections with T. maritimum in sea bream and sea bass for the control of their pathogenic action and also the study of the effect of hydrogen peroxide and chloramine-T, in the control of the disease.The presence of the bacterium T. maritimum was investigated in farmed marine fish in different areas of Greece and the condition of the disease of myxobacteriosis was estimated in Greece, during four (4) seasonal samplings. The presence of myxobacteria in farmed marine fish in Greece, was also described, depending on the geographical origin, the season, the species and the age of fish and also if these strains present a similar biochemical profile, inside Greek borders. Strains of the bacterium T. maritimum, were identified, coded and maintained for future studies.The main objectives of the present thesis are: •Th ...
The present thesis concerns the study of myxobacteria infections from the bacterium Tenacibaculum maritimum in farmed marine fish in Greece and the accomplishment of experimental infections with T. maritimum in sea bream and sea bass for the control of their pathogenic action and also the study of the effect of hydrogen peroxide and chloramine-T, in the control of the disease.The presence of the bacterium T. maritimum was investigated in farmed marine fish in different areas of Greece and the condition of the disease of myxobacteriosis was estimated in Greece, during four (4) seasonal samplings. The presence of myxobacteria in farmed marine fish in Greece, was also described, depending on the geographical origin, the season, the species and the age of fish and also if these strains present a similar biochemical profile, inside Greek borders. Strains of the bacterium T. maritimum, were identified, coded and maintained for future studies.The main objectives of the present thesis are: •The isolation and identification of the pathogens myxobacteria in farmed marine fish in Greece (Mediterranean farmed marine fish) with microbiological, biochemical and molecular methods.•The control of the pathogenic action of the isolated bacteria species in the most sensitive fish species, with experimental infections in sea bream and sea bass.•The histopathological study of the infected fish.•The preservation of the isolated bacteria species for future research, creation of aquaculture myxobacteria bank, from Greek aquaculture farms.The thesis is divided in five Chapters: 1st Introduction, 2nd Material & Methods, 3rd Results, 4th Discussion and 5th Bibliography.In the Introduction, the relevant literature is reviewed. The Chapter is subdivided into three parts. In the first part, the World, European and Greek aquaculture situation is described. In the second part, the pathological problems that are observed in Mediterranean aquaculture are described. In the third part, there is a literature review, in order to collect information for the myxobacteria species that infect farmed marine fish, causing disease. Informations for the disease, the causative agent, the strains, the clinical symptoms, the mechanisms of virulence, the methods of diagnosis, the treatment and the prevention and also the properties of the two antiseptic solutions that are used in the experimental infections, are mentioned. The chapter Material & Methods, is subdivided into five parts. The first part, refers to: The study of the seasonality and the infection rate from the bacterium T. maritinum, in farmed marine fish in Greece. For this reason four (4) seasonal samplings were performed in farmed fish species, Dicentrarchus labrax, Sparus aurata, Pagellus erythrinus, Pargus pargus, Umbrina Cirrosa, Diplodus puntazzo and Sciaena umbra. In every sampling, 200 fish were collected. The bacteria were isolated, identified with microbiological, biochemical and molecular methods, their virulence was controlled and they were coded and storage in deep-freezing. Samples from tissues were also collected for histopathological examinations. The second part, refers to the development of an experimental infection model with the bacterium T. maritimum in sea bream and sea bass, in order to control their pathogenic action.The experimental infections were performed in sea bream and sea bass, with strains of myxobacteria, in certain concentrations, 1.0x106 to 1.5x106cells/ml T. maritimum. The infections were performed with intraperitoneal injection and immersion. The fish were separated in groups, with the appropriate controls respectively. From the experimental infected fish, that showed symptoms and lesions, the causative pathogenic agent that was used, was isolated also, confirming the pathogenicity, while samples from tissues were also collected for histopathological examinations, in order to investigate the lesions of the experimental infected fish.The third part, refers to the effect of the hydrogen peroxide and chloramine-T, in the control of the disease of myxobacteriosis, after experimental infections in sea bream and sea bass. Experimental infections and immersions with the two antiseptic solutions, in experimental infected sea bream and sea bass were performed, in order to control their effectiveness in the treatment of myxobacteriosis.The fourth part, refers to the identification of the Lethal Concentration (LC50) of the hydrogen peroxide and chloramine-T, in sea bream and sea bass.Last, depending on the results from the first experimental infections and immersions, the effectiveness of chloramine-T, was examined in the treatment of infected with myxobacteria juvenile sea bream, in an aquaculture farm. The fifth part, contains the statistical analysis of the results. In the 3rd chapter, the Results are presented.According to the results, was found that T. maritimum, is isolated from the fish, in a percentage of 39.25% (314/800), although during samplings, many fish didn’t show any of the characteristics symptoms and lesions of the disease.The results showed that the majority, 56% (112/200) of the strains of T. maritimum, for all the fish species that were examined, were isolated during the 3rd sampling, in autumn. During the summer period, also there was a slight increase in mortality from spring to the summer. This fact coincided with the parallel development of colonies of Listonella anguillarum, in few fish species, like Shi drum and Brown meagre. The isolation of Listonella anguillarum from Shi drum, from kidney sample was made for the first time. Also, for the first time internationally, T. maritimum was isolated from the cultured fish, Brown meagre (Sciaena umbra).The lowest number of myxobacteria 29% (58/200), was isolated from the fish, during the 4th sampling, in winter.Regarding the infection rate, depending on the fish species, more sensitive appears to be the sea bass with more isolations (128/200) and higher infection rate (64%). The red snapper appears to have the lowest infection rate for the four (4) samplings (18.75%).These results seems to be related with the rise of the water temperature of the sea, because the rate of presence of myxobacteria, raised with the high water temperature, 17ºC-19ºC (112/200 fish). During macroscopic and postmortem examination, the infected fish, regardless of the fish species showed anorexia, sickliness, hemorrhages to the mouth, hemorrhagic stomatitis, pale yellow necrotic lesions on the skin, fins and tail, hyperemia in the flaps, corrosion of the tail and fins. The lesions and symptoms were more severe in the group of fish with body weight 0.5-3gr, regardless of the fish species. Regarding the results of the microbiological examinations, more effective nutrient substrates, proved to be for skin samples, the Flexibacter maritimus medium (FMM) (n=108) and for gills samples, the modified Anacker and Ordal agar (AOA) (n=76). Efforts to isolate myxobacteria from samples taken from kidney or spleen, had no result, while the addition of the antibiotics flumequine and neomycin (in order to have a selective development of the myxobacteria) in the nutrient substrates did not favored the development of the myxobacteria. The true optimum temperature of growth was determined at 19 to 21°C.Regarding the biochemical identification of the strains and the results of API 20E, the main biochemical profile for the 252 of the 314 strains of T. maritimum, was: 000200410. The biochemical profile was similar for all the strains, regardless of the geographical location that were found and the species that were isolated, inside Greek borders. These results show that there is homogeneity in Greece, concerning the biochemical characteristics of T. maritimum. A variation was detected in gelatin, hydrogen sulfide and Voges-Proskauer test, in sixty two strains. The results of the molecular identification and PCR, confirmed that the causative agent in the case of the fish with the lesions and also the healthy fish, was the bacterium T. maritimum.Regarding the infection rate from the monogenea parasites, was proved that the highest infection rate from the parasites, for the seven (7) fish species, was found in autumn (28%), coinciding with the highest infection rate from T. maritimum (56%).The histopathological lesions were found mainly in the gill epithelium and in the skin. The results of the antibiogram for the strains of T. maritimum, showed that the nature of the nutrient substrate affects the size of the inhibition cycle around the antibiotic tablets, while all the strains of T. maritimum showed a similar profile of sensitivity in the antibiotics.In this thesis, 314 strains of T. maritimum isolated and identified from all the fish samples (n=800) and were kept in deep-freezing, -80°C. From the experimental infections that were made with strains of T. maritimum in sea bream and sea bass, was found that the effort of experimental infection with intraperitoneal injection of bacteria, was not effective to cause mortality or the disease to the fish. The prolonged immersion of the fish for 18 hours with a solution of bacteria of T. maritimum proved to be the most effective method of causing the disease of myxobacteriosis. In conclusion, the model of experimental infection of the fish with immersion, in a concentration of bacteria from 1.0x106cells/ml to 1.5x106cells/ml that was described in this thesis, proved to be effective and can constitute the base for future epidemiological studies, in order to prevent and control the disease of myxobacteriosis.The experimental infected sea bass and sea bream with immersion, showed similar lesions with the natural infected fish and high mortality (sea bass 65%, sea bream 35%). These results show clearly the pathogenesis of the isolated strains and also the effectiveness of the experimental immersion to cause the disease to the experimental infected fish. The histopathological lesions were found mainly in the skin and gills and they were similar with the lesions observed in natural infected fish.After the completion of the experimental infections and immersions with hydrogen peroxide and chloramine-T, to the sea bream and sea bass, the fish responded to the therapy, reducing the mortality rate. The immersions were made with: hydrogen peroxide (solution 35%), in the dose 200ppm/30min and chloramine-T, in the dose 12-15ppm/60min.Regarding the sea bream, it was proved that the Lethal Concentration 50 (LC50), for hydrogen peroxide, in 48 h, was 640ppm/30min and for chloramine-T, in 48 h, was the concentration 80ppm/60min.Regarding the sea bass, it was proved that the Lethal Concentration 50 (LC50), for hydrogen peroxide, in 24 h, was 640ppm/30min and for chloramine-T, in 96 h, was the concentration 80ppm/60min.Finally, regarding the application in real conditions, the use of chloramine-T, with 2 immersions at the concentration of 12ppm/60min for the treatment of infected with myxobacteria juvenile sea bream, proved to be very effective.
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