In the sandwich method, negative controls were obtained (1) by the replacement of the primary antibody with another of irrelevant specificity or, (2) omitting the viral suspension step. == 2.3. from duodenum to ileum. In the NV group, no increase in anti-PCV2 PC density was demonstrable in the two sampling instant: the amounts oflamina propriaPCV2-specific antibody-producing PCs remained constant, 10.55 4.24 and 10.06 5.01 at 29 DPI and 53 DPI, respectively. In the V group a significant increase in PCV2-specific antibody-producing PCs was observed over time. The amounts of PCV2-specific antibody-producing PCs increased from 9.37 13.36 at 29 DPI to 18.76 15.83 at 53 DPI. The data on IgA, IgM and IgG PC counts can be considered research values in a populace of adult pigs. The sandwich method can be proposed as a technique able to identify specific antibody-secreting PCs in formalin-fixed paraffin-embedded tissues. A practical application of the sandwich method is the demonstration of a booster-like response of thelamina propriain vaccinated compared to unvaccinated animals. After virus challenge, vaccination induced an increase in the number of PCs made up of specific anti-PCV2 antibodies c-di-AMP at the level of intestinal mucosa. Keywords:Swine, PCV2, Immunity, Vaccination, Intestinal mucosa, Image analysis == 1. Introduction == The gastrointestinal tract’s natural defence mechanisms include the antibacterial properties of gastric and intestinal secretions, a mucus-coated epithelial surface, and the secretion of cytokines and chemokines. The mechanism of intestinal defence that receives most attention, however, is the intestinal immune system, which is usually considered to have the largest accumulation of antibodies in the body (Burkey et al., 2009). Broadly speaking, the organization of the mucosal immune system can be divided into: (a) inductive sites and, (b) effector c-di-AMP sites. Inductive sites are where the uptake of antigens from your mucosal surface and the priming of nave T and B lymphocytes occurs. The other sites recruit several effector mechanisms including the production of secretory immunoglobulin A (SIgA) antibodies (Brandtzaeg et al., 2008). The inductive sites of mucosal immunity include well-defined tissues such as the mucosal-associated lymphoid tissue (MALT) (Liebler-Tenorio and Pabst, 2006) and the loco-regional lymph nodes. The effector sites encompasses different and not well-delimited compartments, such as thelamina propriaof the various mucous membranes that should not be considered part of the MALT (Brandtzaeg et al., 2008). The organization of the immune system in the intestinal mucosa includes nonencapsulated and concentrated lymphoid tissue (Peyer’s patches of the ileum and jejunum) (Burkey et al., 2009) constituting the inductive sites that also include c-di-AMP part of the epithelium associated with the follicles (Sato and Iwasaki, 2005). A diffuse non-encapsulated compartment, including cells (lymphocytes, plasma cells, macrophages, dendritic cells, mast cells) located outside Peyer’s patches but residing in thelamina propria, represents the effector sites where effector mechanisms of cell-mediated and humoral immunity take place (Tizard, 2013). Plasma cells are located mainly round the Rabbit Polyclonal to CDK8 intestinal crypts and form the secretory component of the mucosal immune system, devoted to the production of protective humoral factors acting on the mucosal surface. The intestinal mucosa is usually exposed to heavy loads of commensal and pathogenic microorganisms and since it is the first line of defence, mucosal immunization is considered an interesting target for vaccination (Kim et al., 2012). Therefore the intestinal mucosal immune system has attracted much research interest in recent years. Mucosal responses are more difficult to assess than systemic responses. Mucosal immunity assays can be run through quantifying cell subpopulations (mononuclear cells, neutrophils) or by analyzing immune-related molecules (cytokines, immunoglobulins). There is no gold standard for evaluating secretory immunity in mucosal surfaces. The humoral mucosal immune system can be examined with the same techniques utilized for serology. A critical point is the sampling of secretions, which may be affected by serum or blood contamination, or excessive dilution by other contaminants (ingesta or enzymes) (Guy, 2002). Flushing the mucosal surface does not usually allow the collection of immunoglobulins contained in the mucosal surface film, if the film is usually highly viscous. In these cases, scraping the mucosal surface ensures a more suitable sample for analysis compared to simple flushing, but can increase serum leakage. Because the efficiency of c-di-AMP the intestinal mucosal immune system is useful for diseases where the oro-faecal route is important, c-di-AMP such as the porcine circovirus diseases (PCVDs) (Rose.
