Six reagent waters, eleven diets, and two culture sizes were evaluated for culturing C. dubia. Different filtration techniques were used to prepare the reagent waters. The eleven diets were comprised of two algae augmented with eight supplements. Reproduction and growth were assessed to discern differences among C. dubia raised in mass cultures and cultured in individual cups, during which, bacterial population densities, lipid, protein, and carbohydrate concentrations of the diets were measured. Results showed that a glass-distilled, carbon filtered, deionized reagent water and a Selenastrum capricornutum- Cerophyl® diet were optimum for culturing. Mass culturing supported the highest reproduction and growth, while no correlation was found between nutritional measurements and production.

Both in-situ and ex-situ giant pandas (Ailuropoda melanoleuca), display shifts in bamboo species and part preference throughout the year. The effects of this shifting preference on gastrointestinal (GIT) microbiota were observed using traditional culturing methods to characterize normal GIT microflora from fecal samples and behavioral feeding data of adult male and female pandas over a fourteen-month period. Linear and quadratic fits were used to determine any significant relationships between the time of year and part preference on the GIT microflora (P<0.05). Significant values for time of year were observed with the linear fit in total aerobes (P-value=0.0368), streptococci (P-value=0.0120), and lactobacilli (P-value=0.0166) and quadratic fits in streptococci (P-value=0.0382) and Bacteroides spp. (P-value=0.0134) at á=0.05. Significant linear relationships were observed with part preference and lactobacilli and Bacteroides spp., P-values of 0.0028 and 0.0030, respectively, indicating that part preference and time of year may affect the flux of panda GIT microflora.

Antimicrobial resistance poses a significant challenge to human health and is also a pressing One Health concern. The routine use of antibiotics as growth promoters in agricultural animals has contributed to the emergence of antibiotic resistance, which can subsequently affect human populations. Discontinuing this practice has led to a surge in infections and therapeutic antibiotic use in these animals. This increased susceptibility to infections may be linked, at least partially, to the loss of colonization resistance resulting from alterations in the microbiome. This study focuses on poultry, as the consumption of chicken meat can introduce antibiotic-resistant microbes into the human population. The overarching hypothesis for this research project is that a rationally designed consortium of microbes sourced from healthy chickens will increase colonization resistance and decrease susceptibility to infections as an alternative to growth-promoting antibiotics. The first goal was to analyze the broiler chicken’s gut microbiome and to establish a comprehensive culture collection of microorganisms from healthy chickens. Culture-enriched and culture-independent 16S sequencing was applied to assess the cultivability of the samples and to analyze their microbial profiles. Isolates were identified using MALDI-TOF and 16S rRNA gene sequencing. Frozen samples (from antibiotic-free farms) had a greater microbial diversity than fresh samples (from a university research facility). However, a greater proportion of the microbiome was recovered by culture from the fresh compared to the frozen samples. A strain collection of 1121 isolates representing 121 species was constructed. In Aim 2, I carried out a functional screen to identify isolates from the culture collection that inhibited the growth of the predominant poultry pathogens, E. coli and C. perfringens. Several isolates were identified that inhibited one or the other pathogens and a small number of isolates killed both pathogens. These microbes form the basis of therapeutic consortia to increase colonization resistance in chickens. / Thesis / Master of Science (MSc) / In the poultry industry, antibiotics have been used to promote chicken’s growth. This has contributed to the spread of antibiotic resistance to animal/human pathogens. When the use of growth-promoting antibiotics is stopped, the chickens become more susceptible to infections. These chickens have possibly lost protective bacteria that help fight pathogens. I thought that bacteria from healthy chicken’s intestine could help fight pathogens. To do this, I isolated a large collection of chicken gut’s good bacteria from healthy birds after individually separating them from the mixture using growing methods and sequencing. I separated bacteria from frozen and fresh mixtures, found that more bacteria grow from fresh mixtures. I then tested individual bacteria from this collection to see if they stop pathogenic bacteria like E. coli and C. perfringens from growing. I found that many bacteria could do this which may be used to develop a therapeutic community of good bugs to colonize chickens to make them more disease resistant.

<p>One main hurdle to xenotransplantation, i.e. transplantation between different species, is the immunological barrier that the organ meets in the recipient. The aim of this thesis was to characterise xenogeneic rejection mechanisms by using the concordant mouse-to-rat heart transplantation model.</p><p>Graft-infiltrating immune cells could be isolated from both rejecting and non-rejecting grafts using ex vivo propagation, a technique based on incubation of graft biopsies in culture medium for 48 hours. The numbers of recovered T lymphocytes were considerably higher in grafts undergoing cell-mediated rejection than in grafts undergoing acute vascular rejection (AVR) or in non-rejecting transplants. Thus, ex vivo propagation should be a valuable tool for further studies of cell-mediated rejection.</p><p>Cytokine patterns in the grafts, as measured by a quantitative real-time RT-PCR method, showed that AVR and cell-mediated rejection are associated with an increase of both pro-inflammatory cytokines (IL-1β and TNF-α) and more specific cytokines (IL-2, IL-10, IL-12p40 and IFN-γ). These data differed considerably from the patterns seen in the spleens of the recipients. Cell-mediated xenograft rejection was also found to be associated with a local accumulation of hyaluronan.</p><p>Oral administration of xenogeneic cells stimulated a production of antibodies that could induce hyperacute rejection of cardiac xenografts when passively transferred to graft recipients. This is in contrast to several models for autoimmune diseases and allogeneic transplantation where oral administration of antigens is an effective way to induce unresponsiveness. Hence, future attempts to induce oral tolerance in xenotransplantation should be done with caution.</p>

One main hurdle to xenotransplantation, i.e. transplantation between different species, is the immunological barrier that the organ meets in the recipient. The aim of this thesis was to characterise xenogeneic rejection mechanisms by using the concordant mouse-to-rat heart transplantation model. Graft-infiltrating immune cells could be isolated from both rejecting and non-rejecting grafts using ex vivo propagation, a technique based on incubation of graft biopsies in culture medium for 48 hours. The numbers of recovered T lymphocytes were considerably higher in grafts undergoing cell-mediated rejection than in grafts undergoing acute vascular rejection (AVR) or in non-rejecting transplants. Thus, ex vivo propagation should be a valuable tool for further studies of cell-mediated rejection. Cytokine patterns in the grafts, as measured by a quantitative real-time RT-PCR method, showed that AVR and cell-mediated rejection are associated with an increase of both pro-inflammatory cytokines (IL-1β and TNF-α) and more specific cytokines (IL-2, IL-10, IL-12p40 and IFN-γ). These data differed considerably from the patterns seen in the spleens of the recipients. Cell-mediated xenograft rejection was also found to be associated with a local accumulation of hyaluronan. Oral administration of xenogeneic cells stimulated a production of antibodies that could induce hyperacute rejection of cardiac xenografts when passively transferred to graft recipients. This is in contrast to several models for autoimmune diseases and allogeneic transplantation where oral administration of antigens is an effective way to induce unresponsiveness. Hence, future attempts to induce oral tolerance in xenotransplantation should be done with caution.

From 2006-2011, four population reintroduction techniques were applied to three sites within a reach of the upper Clinch River in Virginia designated suitable for population restoration of the federally endangered oyster mussel (Epioblasma capsaeformis). These techniques were: 1) translocation of adults (Site 1), 2) release of laboratory-propagated sub-adults (Site 1), 3) release of 8-week old laboratory-propagated juveniles (Site 2), and 4) release of stream-side infested host fishes (Site 3). Demographic data were collected in 2011 and 2012 by systematic quadrat and capture-mark-recapture sampling to assess reintroduction success, evaluate reintroduction techniques, and compare survey approaches for monitoring freshwater mussels. Estimates of abundance and density of translocated adults ranged from 450-577 individuals and 0.09-0.11/m2 in 2011, and 371-645 individuals and 0.07-0.13/m2 in 2012. Estimates of abundance and density of laboratory-propagated sub-adults ranged from 1,678-1,943 individuals and 0.33-0.38/m2 in 2011, and 1,389-1,700 individuals and 0.27-0.33/m2 in 2012. Additionally, three recruits were collected at Site 1. No E. capsaeformis were collected at Sites 2 and 3. Capture-mark-recapture sampling produced similar mean point estimates as systematic quadrat sampling, but with typically more precision. My results indicated that the release of larger individuals (>10 mm) is the most effective technique for restoring populations of E. capsaeformis, and that systematic quadrat and capture-mark-recapture sampling have useful applications in population monitoring that are dependent on project objectives. Systematic quadrat sampling is recommended when the objective is to simply estimate and detect trends in population size for species of moderate to larger densities (>0.2/m2). Capture-mark-recapture sampling should be used when objectives include assessing a reintroduced population of endangered species or at low density, obtaining precise estimates of population demographic parameters, or estimating population size for established species of low to moderate density (0.1-0.2/m2). The ability to grow endangered juveniles to larger sizes in captivity requires improving grow-out culture methods of laboratory-propagated individuals. A laboratory experiment was conducted to investigate the effects of temperature (20-28 C) on growth and survival of laboratory-propagated juveniles of the Cumberlandian combshell (Epioblasma brevidens), E. capsaeformis, and the wavyrayed lampmussel (Lampsilis fasciola) in captivity. Results indicated that 26 C is the optimum temperature to maximize growth of laboratory-propagated juveniles in small water-recirculating aquaculture systems. Growing endangered juveniles to larger sizes will improve survival in captivity and after release into the wild. As a result, hatcheries can reduce the time that juveniles spend in captivity and thus increase their overall production and enhance the likelihood of success of mussel population recovery efforts by federal and state agencies, and other partners. / Master of Science

No description available.

The mesenchymal stromal cell (MSC) is of great interest for its immunomodulatory and regenerative properties. However, to research and use these MSCs it is essential to identify and characterize them as such. They need to fulfill the MSCs' minimal criteria which assess the differentiation potential, cell surface markers, and adherence. In this study, cells donated from human bone marrow were identified as MSC according to the minimal criteria. Methods used were flow cytometry, immunofluorescent staining, and ELISA. Furthermore, the population was cultured in three different media (DMEM-LG with either 10% FBS, 2% FBS, or 10% FBS supplemented with 10% conditioned media from human urinary bladder carcinoma cells (T24)) for 21 days whereupon tested for the mesenchymal characteristics, cells were counted and size measured at every passage. All cultures maintained their mesenchymal character, however, cells grown in 2% FBS became a considerably more heterogenous population regarding cell size and granularity, perhaps because of senescence. Additionally, these cells somewhat decreased in proliferation and resulted in 1 x 106 cells after 21 days, however, this was not a significant decrease when compared to the 10% FBS culture which had 2.16 x 106 cells after 21 days (p=0.061). On the contrary, the culture supplemented with T24 conditioned media resulted in a significantly higher cell count with 4.75 x 106 cells (p=0.008). Further studies could investigate which components in the conditioned media contributed to the proliferation. Moreover, the cell population in this study could not be characterized as MSC with certainty as additional cell surface markers should be tested.

No description available.

No description available.