Coccidiosis has a negative economic impact on the commercial poultry industry, and probiotics are beneficial bacteria that aid in maintaining healthy gut microflora. We hypothesized that probiotic administration would positively affect gut morphology and increase IgG secretion during an Eimeria challenge, which was evaluated by measuring total chicken IgG and gut morphology (villus height, villus width, villus surface area, crypt depth, villus height to crypt depth ratio and lamina propria thickness). On day-of-hatch, broilers were placed into floor pens with 50 percent pine shavings and 50 percent used litter. The broilers were exposed to Eimeria oocysts via the feed on day 14 and challenged on day 36. On days 6, 22, 36, and 43, tissue samples from the intestine were collected for morphological evaluation, and blood samples were taken to quantify chicken IgG from serum. Data were measured using a factorial ANOVA and main effect means were deemed significant at P ≤ 0.05. In cases where significant interactions were observed, data was subjected to a one-way ANOVA. All means were separated using a Duncan’s Multiple Range Test. On day 6 in the duodenum, a significant interaction was observed regarding vaccination and probiotic administration (Coccivac®-B, Intervet/Schlering-Plough Animal Health/Merck and Co., Inc., Whitehouse Station, NJ). Villus height to crypt depth ratio decreased in ionophore treated birds compared to control birds in the duodenum and lower ileum on day 6, 36, and 43. Villus crypt depth in vaccinated birds decreased in the duodenum after the challenge. On day 43, the ionophore treated birds had less villus height and surface area compared to control and vaccinated birds, while lamina propria thickness increased in the duodenum, and non probiotic birds had longer villi than probiotic birds. On day 22, vaccinated birds had significantly increased chicken IgG levels compared to the control and ionophore birds, and the non probiotic birds had significantly increased IgG secretion compared to probiotic fed birds. On day 36, the ionophore birds had significantly increased levels of IgG compared to the control birds, which could also support that the ionophore delayed exposure to the parasite. These results suggest that gut morphology and humoral immunity are affected by probiotic administration, coccidiosis vaccination, ionophore application and Eimeria challenge. Both the day 43 morphology results and day 36 chicken IgG results for the ionophore treated birds demonstrates that ionophore administration delays exposure of the avian gut to invasive coccidia. More research is necessary to evaluate how probiotics influence coccidiosis vaccination and humoral immunity, so that probiotics may be used to improve the effectiveness of coccidiosis vaccination and to evaluate if probiotics aid in ameliorating the effects of an Eimeria infection.

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Thesis (M.S.)--Northern Michigan University, 2009. / Includes bibliographical references (leaves 65-73).

The immobilization of antibodies on silica surfaces has been a wide and common practice via cross-linking with the formation of covalent bonds between surface and antibody. The formation of antibody thin films on solid surfaces using pneumatic spray (PS) as the deposition technique and the analysis of the surface morphology of these films were investigated during this study. The pneumatic spray method was compared with the covalent bonding method Avidin-Biotin Bridge (ABB). The intensities and capture efficiency tests showed similar results for both techniques with a lower signal-to-noise ratio (SNR) for the PS deposited films. Specificity tests suggested that the bio-sensitivity of the antibody films that were pneumatically sprayed maintained their capture abilities after the immobilization process. Analysis obtained from the attenuated total reflectance Fourier transform infrared ATR-FTIR support these results indicating that the antibodies retained their native structure and chemical stability thorough the induced physisorption process. The pneumatic spray films also preserve mechanical stability by adhering to the surface after the rinsing procedures. Capture efficiency was tested for both immobilization techniques, the results of which were similar. The pneumatic spray technique was also tested using a diverse range of deposition times. It was shown that a 2 minute deposition time was sufficient to produce a film with similar capture efficiency to the avidin-biotin bridge technique. The surface density obtained for the 2 minute deposition was 9.05 ng/mm2, which is higher than the range of 2.2 to 4.74 ng/mm2 reported for the avidin-biotin bridge technique[1-3]. The contact angle measurements for the pneumatic spray films showed a higher hydrophobicity compared with the avidin-biotin bridge films. This is due to the higher surface roughness obtained for the avidin-biotin bridge films, a higher surface density for the PS and the random orientation of the antibodies in the pneumatic spray films. A study of shelf life showed that the pneumatic spray technique produces stable films that can be used for as long 100 days (study performed only up to 100 days) with similar capture efficiency to those prepared in the same day. To further understand the improvement in capture efficiency of the pneumatic spray films, the surface morphology was investigated to determine its influence in the cell adhesion process. The surface was characterized by several different techniques: ellipsometry to determine the thickness of the films, atomic force microscopy (AFM) to calculate the surface roughness, optical microscopy to identify particle formation during antibody immobilization process, fluorescent microscopy and sandwich fluorescent immunoassay to observe the immobilization patterns of antibodies and antigens on the surface, contact angle measurements to analyze the wettability of the antibody films and X-ray photoelectron spectroscopy (XPS) to confirm the presence of antibody on both deposition methods and to propose a growth model for the pneumatic spray deposition technique. A possible explanation for the similar results of capture efficiency for both techniques can be attributed to three main factors. First, the antibodies retained their native structure thorough the induced physisorption process allowing then to capture antigen normally. Second, the lack of orientation of the antibodies in the pneumatic films was compensated by high surface density thereby offering more binding sites to capture antigens. Third, hydrophobic surfaces are favorable to cell adhesion, therefore the high hydrophobicity of the pneumatic spray films increases the capture efficiency. It is important to mention that the time that it takes to produce the immunoassay surfaces was reduced dramatically from more than twenty four hours for the avidin-biotin bridge films to only a few minutes for the pneumatic spray films. In addition, pneumatic spray films significantly reduce the amount of materials and chemicals used in the deposition process. These factors make the pneumatic spray technique an excellent technique for the immobilization of antibodies on glass slides for commercial bio-sensor devices.

Iron is an essential element for virtually all forms of life. Complicating matters, it is present in the insoluble ferric form in aerobic environments, while the more soluble ferrous form is found in anaerobic or reducing environments. Vibrio cholerae, the causative agent of the disease cholera, requires iron to survive. In order to meet the need for iron, V. cholerae expresses a variety of iron acquisition systems. One of these systems, Feo, is highly conserved among bacterial species as well as archaea and transports ferrous iron. The Feo system consists of three proteins: FeoA, FeoB, and FeoC. Previous work using the bacterial adenylate cyclase two hybrid system has shown that FeoC interacts with the cytoplasmic N-terminal domain of FeoB. However, the significance of this interaction is not known. In this study, V. cholerae Feo system proteins were analyzed for residues important for the interaction between FeoB and FeoC. In addition, FeoA and FeoC were purified for antibody production. It was found that a residue in the G protein domain of FeoB was not necessary for interaction with FeoC. However, a conserved residue in FeoC did abolish the interaction with FeoB. These results indicate that there is at least one residue important in the interaction of FeoB and FeoC, although further characterization will most likely reveal more. Antibodies to FeoA and FeoC were generated to use them for further characterization of the Feo system. / text

Therapeutic antibodies have achieved exceptional clinical success in the treatment of cancer and other human diseases. Now, new approaches are required to enhance the potency of antibodies to further increase the number of patients responding to therapy. By engineering the antibody Fc domain through mutation of the amino acid sequence, binding affinity to activating or inhibitory Fc receptors on effector cells can be increased to modulate the cellular immune response. However, attaining selectivity for closely related Fc receptors has proved challenging and the technique has not been applied to access the function of antibody isotypes other than IgG. Here we present new methods for enhancing antibody potency using both hybrid IgA/G and aglycosylated Fc domains. In the first instance, a chimeric antibody Fc domain has been created by combining residues from IgA with those from IgG. The new variant, MutD, introduces binding to FcαRI while retaining affinity for certain members of the FcγR family. ADCC assays show MutD, when part of a full length trastuzumab antibody against Her2 antigen, can kill Her2-overexpressing tumor cell lines as effectively as IgA antibodies. Moreover, MutD shows improved assembly compared to IgA and thus provides access to potent FcαRI function while overcoming the expression and purification barriers that have limited the use of IgA as a therapeutic. Alternatively, aglycosylated antibodies may be engineered for exceptional effector function. Glycans anchored to residue N297 of the antibody IgG Fc domain are typically critical in mediating binding toward the FcγRs. Yet, using a full length bacterial IgG display system, we have isolated aglycosylated Fc1004 with mutations that confer a 160-fold increase in the affinity toward the low affinity FcγRIIa-R131 allele as well as high selectivity against binding to the remarkably homologous inhibitory receptor, FcγRIIb. Incorporation of this engineered Fc into trastuzumab resulted in a 75% increase in tumor cell phagocytosis by macrophages compared to that of the parental glycosylated trastuzumab with medium Her2-expressing cancer cells. In vivo testing of Fc1004 using NOD/SCID mouse model, reconstituted by adoptive transfer of leukocytes from FcγRIIa-R131 homozygous donors, showed a promising reduction in tumor burden in SkBr-3 Her2+ xenografts. / text

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / abstract / toc / Biochemistry / Doctoral / Doctor of Philosophy

No description available.