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Metodické přístupy k vyšetření lymfoidních tkání jeseterů
ŠÁLKOVÁ, Eva
We present material, data and results that were encountered in doctoral study and summarized in dissertation thesis. Our research was focused on methodical approaches to examination of lymphoid tissues in sturgeons. Sturgeons live on Earth for a long period of time. Well-developed immune system enabled sturgeons to face and deal with different attacks, and thus played an important and crucial role in sturgeon survival until present days. Sturgeons exhibit large genomes among fishes and are well known for their polyploidy origin. Acipenseriformes have a high tolerance for hybridization as well as for spontaneous doubling of chromosome sets. In their evolution, at least three independent polyploidization events have taken place, in the literature known as 3R hypothesis (three rounds hypothesis). Therefore, the present research focused on the haematopoietic and lymphoid tissues examination. Representatives of different ploidy level groups were encountered in the study: sterlet (Acipenser ruthenus) represented diploid (2n) group, Russian sturgeon (Acipenser gueldenstaedtii) was chosen as tetraploid group representative (4n), and finally shortnose sturgeon (Acipenser brevirostrum) served as hexaploid group representative (6n). Haematopoietic and lymphoid tissues were conventionally histologically processed, obtained sections were stained using haematoxylin and eosin. Advanced histological staining methods were applied when examining tissues to map and recognize particular cell types involved in haemato- and lymphopoiesis. Hassall's corpuscles were detected in thymi, particular in the thymic medulla; to our knowledge, it was the first description of Hassall's corpuscles in sturgeons. Subsequently, the thymus of juvenile sterlet (Acipenser ruthenus) was examined immunohistochemically. The wide range cytokeratin, vimentin, S-100 protein, leukocyte common antigen LCA (CD45) and CD3 were selected as the markers to map the thymic microenvironment. Commercially available human antibodies were applied on fish tissue, and wide range cytokeratin, vimentin, S-100 protein brought conclusive results and distinguished nature of different microenvironmental components. Contrarily, LCA and CD3 application was negative or inconclusive, and failed. Study confirmed the possibility of human antibody application on fish tissue, together with the need to generate fish and species specific antibodies, mostly when examining lymphoid tissues. Except for the histological and immunohistochemical examination, respectively, the lymphoid or immune system should be tested when using the determination of white blood cell parameters. Generally, the blood is more easily accessible (vessel puncture), and the determination of the total number of leukocytes and the differential counts represent important fish health and physiology status. Under the study, representatives of three different ploidy level groups were tested and white blood cell parameters were monitored monthly during a period of one year. Data suggested the significant effect of ploidy level on total number of leukocytes and morphological nuclear changes in granulocytes and lymphocytes. Annual variation in differential leukocyte counts depended on the species and influence of various external conditions rather than ploidy level. Examination of white blood cell parameters (total number of leukocyte and differential count), as well as histological and immunohistochemical examination, respectively, represent the main methodical approaches for lymphoid organs and immune system assessment in sturgeons. Methods should be applied in an ichthyological practise. Obtained results contribute to fish health status evaluation.
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