Quantification of equine immunoglobulin A in serum and secretions by a fluorescent bead-based assay.

Only few quantitative reports exist about the concentrations and induction of immunoglobulin A (IgA) in mucosal secretions of horses. Despite this, it is widely assumed that IgA is the predominant immunoglobulin on mucosal surfaces in the horse. Here, two new monoclonal antibodies (mAbs) against equine IgA, clones 84-1 and 161-1, were developed and characterized in detail. Both IgA mAbs specifically bound monomeric and dimeric equine IgA in different applications, such as Western blots and fluorescent bead-based assays. Cross-reactivity with other equine immunoglobulin isotypes was not observed. The new IgA mAb 84-1 was used in combination with the previously characterized anti-equine IgA mAb BVS2 for the development and validation of a fluorescent bead-based assay to quantify total IgA in equine serum and various secretions. The IgA assay's linear detection ranged from 64pg/ml to 1000ng/ml. For the quantification of IgA in serum or in secretions an IgA standard was purified from serum or nasal wash fluid (secretory IgA), respectively. The different standards were needed for accurate IgA quantification in the respective samples taking the different signal intensities of monomeric and dimeric IgA on the florescent bead-based assay into account. IgA was quantified by the bead-based assay established here in different equine samples of healthy adult individuals. In serum the median total IgA was 0.45mg/ml for Thoroughbred horses (TB, n=10) and 1.16mg/ml in Icelandic horses (ICH, n=12). In nasopharyngeal secretions of TB (n=7) 0.13mg/ml median total IgA was measured, and 0.25mg/ml for ICH (n=12). Saliva of ICH (n=6) contained a median of 0.15mg/ml, colostrum of Warmbloods (n=8) a median of 1.89mg/ml IgA. Compared to IgG1 and IgG4/7 quantified in the same samples, IgA appeared as the major immunoglobulin isotype in nasopharyngeal secretions and saliva while it is a minor isotype in serum and colostrum. The newly developed monoclonal antibodies against equine IgA and the resulting bead-based assay for quantification of total IgA can notably improve the evaluation of mucosal immunity in horses.

Maternal T-lymphocytes in equine colostrum express a primarily inflammatory phenotype.

The purpose of this study was to characterize maternal immune cells in colostrum of mares. Cell phenotypes and cytokine secretion from mare peripheral blood mononuclear cells (PBMC) and cells from colostrum were analyzed by flow cytometry and by multiplex cytokine analysis. Equine colostral leukocytes were composed of mainly CD8(+) and CD4(+) lymphocytes. CD8(+) cells were significantly enriched in colostrum compared to PBMC (n=35). Colostral T-cells (n=13) responded to stimulation with PMA/ionomycin with a significantly higher magnitude of IL-17 (p=0.037) and similar IFN-γ concentrations (p=0.305), while IL-4 (p=0.0002) and IL-10 (p=0.0002) production was decreased compared to PBMC. CD4(+) and CD8(+) T-cells in colostrum produced IFN-γ (n=4). The findings show that colostrum T-cells can produce all four cytokines investigated here but most cells are polarized toward IL-17 and IFN-γ production and an inflammatory phenotype. Maternal T-cells likely migrate to the colostrum in a selective manner and may have specific roles in neonatal immune development.

Occurrence of IgE in foals: evidence for transfer of maternal IgE by the colostrum and late onset of endogenous IgE production in the horse.

IgE is the key antibody involved in type I allergies. Allergen mediated crosslinking of IgE bound to high affinity Fcepsilon-receptors on mast cells and basophils stimulates cellular degranulation and release of inflammatory mediators and cytokines. In this report, we demonstrate that IgE antibodies can be transferred from the mother to offspring in horses via the colostrum. We found a clear correlation between the IgE concentration in colostrum and the total IgE concentration in foal sera on day 2 after birth (r(sp)=0.83). Maternal IgE was detected in foal sera by ELISA and on peripheral blood leukocytes of foals by flow cytometry. Both serum and cell membrane-bound IgE were undetectable in newborn foals before colostrum uptake and peaked on days 2-5 after birth. Cell-bound IgE became undetectable at 2 months after birth. Serum IgE disappeared from the circulation within the first 3-4 months of age. These kinetics suggest that the IgE antibodies which are detectable in foals during the first 4 months after birth are of maternal origin only. The endogenous IgE production was found to begin at 9-11 months of age, when IgE could be detected on peripheral blood leukocytes and in foal sera again. After 18 months of life, the total IgE concentrations in foal sera were comparable to those detected in their dams. The late onset of endogenous IgE production offers an explanation for observations that IgE mediated allergies are generally not observed in horses before puberty. The roles of the passively transferred maternal IgE in newborn foals are not yet known, but could be manifold, ranging from passive immunity and induction of immunoregulatory functions to determinative influences of maternal IgE on the antibody repertoire in the offspring.