In the thesis a comparative analysis and justification of epizootic risks associated with reptiles and amphibians is presented. The assessment of the methods of preparing primary cell lines of poikilotermic animals and comparative analysis of culture conditions for these cells is made. Also here we report for their sensitivity to several mammalian viruses and use of reptilian cell culture in attempts to isolate the putative etiologic virus of reptiles with respiratory pathology. Reptilian cell cultures are rare and largely derived from sea turtles affected by tumors. To our knowledge, this thesis is the first to describe primary cell cultures obtained from normal tissues of reptile and amphibian in Ukraine, and the first report to describe established cell culture from tissue of Red eared slider (Trachemis scripta elegance). Here we describe efficient establishment of primary cell cultures from tissue of Amphibian and Reptile. Primary cell cultures were obtained from parenhimal organs of African clawed frog Xenopus laevis (XL) and Red-eared slider, Trachemys scripta elegance (TF). And primary cell culture of dropped tail Sand lizard, Lacerta agilis (LA) was obtained without animal sacrificing. The primary cell cultures were established by different methods of fermentative disaggregation and tissue explants method was used also. The method of fractional heat trypsinization (at 37 °C) as a best method for reptile tissues disaggregation was selected. The method of fractional trypsinization without heating (at 18 - 22) was chosen as an optimal for primary cell culture obtaining from amphibian tissue. It has been determined an optimal temperature conditions, medium and cell surfaces and for each cell culture. All three cell cultures grew optimally at temperature near 29 °C, but experienced loss viability when cultured at 37 °C. Acceptable growth of all cells occurred in some of standard cell culture media, such as DMEM, RPMI-1640 and their composition, but amphibian cells XL required osmotic pressure correction (from 290-300 mOsm/L to180-250 mOsm/L, as an osmotic pressure of amphibians' plasma). All obtained cell cultures required a FBS for proliferation. The growth surface coating with FBS was the most cost- and time-effective and the most promoted cell adhesive and proliferation. During propagation reptile and amphibian cell cultures exhibited a fibroblast-like or mixed morphology. The XL and LA cell cultures have been subcultured up to eight times before signs of senescence and crisis of cell culture have been seen. The XL and LA were determined as a finite cell culture. The different methods of maintaining/increasing proliferation and immortalization of cell cultures were investigated (increasing FBS level in culture media up to 20%, using of conditioned culture media from continuous cell lines after sterilization per 0,22 µm filter, combination of these methods and additition of Actovegin at 224 µg/ml). Utilization of Actovegin at 224 µg/ml culture media led to increasing proliferation, stabilization and immortalization TF cell culture. The TF cell culture has been subcultured for more than 100 times successfully by the time of the thesis writing.
The established cell line TF, from Red-eared slider (Trachemys scripta elegance) tissue, has been obtained for the first time. Here we describe full characterization of established cell line TF, from Red-eared slider (Trachemys scripta elegance) tissue, included validation fibroblast cells by morphology and immunocytochemistry, and confirmation species-specificity by chromosomal analysis. The TF cells were fibroblast like, and were stained by Pappenheym method to morphology detailsation and were stained with antibody against vimentin, a type III intermediate filaments, known as a reliable fibroblast marker. Chromosomal analysis using 69 passaging level cells revealed that TF cells had normal diploid chromosome number of 2n=50 and chromosome number and morphology confirmed belonging of cells to the Trachemys scripta elegance species.
The TF cell line susceptibilities to warm-blooded animal viruses were investigated. The Aueski desease virus (Herpesviridae), Newcastle desease virus (Paramyxoviridae) and vesicular stomatitis virus (Rhabdoviridae) propagated in the cell line, causing nonspecific CPE. According to three times passaging results TF cells were highly capable of being successfully infected with these viruses. The titres of these viruses on 48 h after inoculation were 3,7 ± 0,07 lg ТЦД50/см3 for Aueski desease virus, 5,8 ± 0,12 lg ТЦД50/см3 for vesicular stomatitis virus and 7,1 ± 0,09 lg ТЦД50/см3 for Newcastle desease virus. Our established cell line was suitable for unknown virus isolation from tissues of ill dead reptiles Ouroborus cataphractus, Furcifer pardalis and Psammobates geometricus. This conclusion was based on the CPE presence in TF cell culture. The virus was able to replicate in reptilian cell line TF at 29 °С, and was not able at 37 °
