The bursa of Fabricius is a central lymphoid organ essential for B cell development and generation of humoral immune responses in birds. The bursa is connected dorsally to the cloaca and continually “samples” the intestinal fluid phase for the presence of antigen. It is comprised of folds or plicae, which are seeded with individual follicles, where antigen processing and presentation occurs for B cell development as well as generation of antibody responses. The plicae are separated from the bursal lumen by interfollicular epithelium (IFE) and specific regions of follicle-associated epithelium (FAE). The FAE is comprised of M cell-like cells, which are specialized for transcytosis of antigen from the bursal lumen into underlying follicles. The uptake of particulate and soluble antigen in the bursa of Fabricius has been previously demonstrated, but how particle size affects their internalization within bursal FAE and the transport of particulate antigen into deeper follicles has not been explored. It has been shown that nanoparticles (NPs) ≤40 nm are most efficiently internalized by the epithelium of the murine intestine and vaginal tract. Therefore, we examined the uptake of 0.04 - 2 μm fluorescent polystyrene NPs in bursa at 1 hour or 6 hours after bursal administration to determine whether bursal epithelium is similarly constrained. Using immunofluorescence microscopy (IFM) and spectrofluorophotometry we found that NP uptake is inversely correlated with NP size. NPs ≤40 nm are most efficiently internalized by the bursal epithelium and bursal follicles, while NPs ≥500 nm are not effectively taken up by the bursal epithelium within 6 hours of administration. Moreover, once the size-limited capabilities of the bursal epithelium were established, we also found that bursal priming of chickens with 20 nm NPs conjugated to IMP-1, a protective antigen of an important avian pathogen Eimeria maxima, induces IMP-1-specific serum IgG following sub-cutandous boosting. We induced similar IMP-1-specific serum antibody responses in chickens primed bursally and sub-cutaneously boosted as those primed and boosted sub-cutaneously. Whether this route of immunization is able to elicit long-term protection must be investigated. A number of infectious diseases, including Infectious Bursal Disease Virus (IBDV), which directly targets the bursa of young birds and prevents the development of the immune system and causes mortality, are prevalent in the poultry industry. While vaccines exist for many of these diseases they confer only partial or incomplete protection; therefore, alternative vaccine strategies must be investigated. In addition, the bursa is an ideal in vivo model for the investigation of endocytic mechanisms for uptake of particulate antigen. Therefore, further characterizing the mechanisms of NP uptake at mucosal surfaces and their immunogenicity will be important for the development of NP-based mucosal vaccines for agriculturally relevant species such as poultry.

Confocal Raman Microscopy, a non-invasive, non-destructive and label-free technique, was employed to study the uptake and localization of nanoparticles (NPs) in the Hepatocarcinoma human cell line HepG2 at the level of single cells. Cells were exposed to carbon nanotubes (CNTs) the surface of which was engineered with polyelectrolytes and lipid layers, aluminium oxide and cerium dioxide nanoparticles. Raman spectra deconvolution was applied to obtain the spatial distributions of NPs together with lipids/proteins in cells. The colocalization of the NPs with different intracellular environments, lipid bodies, protein and DNA, was inferred. Lipid coated CNTs associated preferentially with lipid rich regions, whereas polyelectrolyte coated CNTs were excluded from lipid rich regions. Al2O3 NPs were found in the cytoplasm. CeO2 NPs were readily taken up and have been observed all over the cell. Raman z-scans proved the intracellular distribution of the respective NPs.