Transcriptome analysis of immune genes in peripheral blood mononuclear cells of young foals and adult horses.

During the neonatal period, the ability to generate immune effector and memory responses to vaccines or pathogens is often questioned. This study was undertaken to obtain a global view of the natural differences in the expression of immune genes early in life. Our hypothesis was that transcriptome analyses of peripheral blood mononuclear cells (PBMCs) of foals (on day 1 and day 42 after birth) and adult horses would show differential gene expression profiles that characterize natural immune processes. Gene ontology enrichment analysis provided assessment of biological processes affected by age, and a list of 897 genes with ≥2 fold higher (p<0.01) expression in day 42 when compared to day 1 foal samples. Up-regulated genes included B cell and T cell receptor diversity genes; DNA replication enzymes; natural killer cell receptors; granzyme B and perforin; complement receptors; immunomodulatory receptors; cell adhesion molecules; and cytokines/chemokines and their receptors. The list of 1,383 genes that had higher (p<0.01) expression on day 1 when compared to day 42 foal samples was populated by genes with roles in innate immunity such as antimicrobial proteins; pathogen recognition receptors; cytokines/chemokines and their receptors; cell adhesion molecules; co-stimulatory molecules; and T cell receptor delta chain. Within the 742 genes with increased expression between day 42 foal and adult samples, B cell immunity was the main biological process (p = 2.4E-04). Novel data on markedly low (p<0.0001) TLR3 gene expression, and high (p≤0.01) expression of IL27, IL13RA1, IREM-1, SIRL-1, and SIRPα on day 1 compared to day 42 foal samples point out potential mechanisms of increased susceptibility to pathogens in early life. The results portray a progression from innate immune gene expression predominance early in life to adaptive immune gene expression increasing with age with a putative overlay of immune suppressing genes in the neonatal phase. These results provide insight to the unique attributes of the equine neonatal and young immune system, and offer many avenues of future investigation.

Antigen-specific immunoglobulin variable region sequencing measures humoral immune response to vaccination in the equine neonate.

The value of prophylactic neonatal vaccination is challenged by the interference of passively transferred maternal antibodies and immune competence at birth. Taken our previous studies on equine B cell ontogeny, we hypothesized that the equine neonate generates a diverse immunoglobulin repertoire in response to vaccination, independently of circulating maternal antibodies. In this study, equine neonates were vaccinated with 3 doses of keyhole limpet hemocyanin (KLH) or equine influenza vaccine, and humoral immune responses were assessed using antigen-specific serum antibodies and B cell Ig variable region sequencing. An increase (p<0.0001) in serum KLH-specific IgG level was measured between days 21 and days 28, 35 and 42 in vaccinated foals from non-vaccinated mares. In vaccinated foals from vaccinated mares, serum KLH-specific IgG levels tended to increase at day 42 (p = 0.07). In contrast, serum influenza-specific IgG levels rapidly decreased (p≤0.05) in vaccinated foals from vaccinated mares within the study period. Nevertheless, IGHM and IGHG sequences were detected in KLH- and influenza- sorted B cells of vaccinated foals, independently of maternal vaccination status. Immunoglobulin nucleotide germline identity, IGHV gene usage and CDR length of antigen-specific IGHG sequences in B cells of vaccinated foals revealed a diverse immunoglobulin repertoire with isotype switching that was comparable between groups and to vaccinated mares. The low expression of CD27 memory marker in antigen-specific B cells, and of cytokines in peripheral blood mononuclear cells upon in vitro immunogen stimulation indicated limited lymphocyte population expansion in response to vaccine during the study period.

Immunogenicity of an electron beam inactivated Rhodococcus equi vaccine in neonatal foals.

Rhodococcus equi is an important pathogen of foals that causes severe pneumonia. To date, there is no licensed vaccine effective against R. equi pneumonia of foals. The objectives of our study were to develop an electron beam (eBeam) inactivated vaccine against R. equi and evaluate its immunogenicity. A dose of eBeam irradiation that inactivated replication of R. equi while maintaining outer cell wall integrity was identified. Enteral administration of eBeam inactivated R. equi increased interferon-γ production by peripheral blood mononuclear cells in response to stimulation with virulent R. equi and generated naso-pharyngeal R. equi-specific IgA in newborn foals. Our results indicate that eBeam irradiated R. equi administered enterally produce cell-mediated and upper respiratory mucosal immune responses, in the face of passively transferred maternal antibodies, similar to those produced in response to enteral administration of live organisms (a strategy which previously has been documented to protect foals against intrabronchial infection with virulent R. equi). No evidence of adverse effects was noted among vaccinated foals.

Effects of opsonization of Rhodococcus equi on bacterial viability and phagocyte activation.

OBJECTIVE: To investigate the effect of opsonization of Rhodococcus equi with R. equi-specific antibodies in plasma on bacterial viability and phagocyte activation in a cell culture model of infection. SAMPLE: Neutrophils and monocyte-derived macrophages from 6 healthy 1-week-old foals and 1 adult horse. PROCEDURES: Foal and adult horse phagocytes were incubated with either opsonized or nonopsonized bacteria. Opsonization was achieved by use of plasma containing high or low concentrations of R. equi-specific antibodies. Phagocyte oxidative burst activity was measured by use of flow cytometry, and macrophage tumor necrosis factor (TNF)-α production was measured via an ELISA. Extracellular and intracellular bacterial viability was measured with a novel R. equi-luciferase construct that used a luminometer. RESULTS: Opsonized bacteria increased oxidative burst activity in adult horse phagocytes, and neutrophil activity was dependent on the concentration of specific antibody. Secretion of TNF-α was higher in macrophages infected with opsonized bacteria. Opsonization had no significant effect on bacterial viability in macrophages; however, extracellular bacterial viability was decreased in broth containing plasma with R. equi-specific antibodies, compared with viability in broth alone. CONCLUSIONS AND CLINICAL RELEVANCE: The use of plasma enriched with specific antibodies for the opsonization of R. equi increased the activation of phagocytes and decreased bacterial viability in the extracellular space. Although opsonized R. equi increased TNF-α secretion and oxidative burst in macrophages, additional factors may be necessary for effective intracellular bacterial killing. These data have suggested a possible role of plasma antibody in protection of foals from R. equi pneumonia.