Angela I. Bordin

Activation of foal neutrophils at different ages by CpG oligodeoxynucleotides and Rhodococcus equi.

Toll-like receptor 9 (TLR9) activation stimulates protective immune responses against intracellular pathogens by phagocytes, including neutrophils. This study examined TLR9-mediated neutrophil activation in neonatal foals. Unmethylated CpGs, ligands for TLR9, were used to stimulate equine neutrophils, either purified or in contact with other peripheral blood leukocytes. Rhodococcus equi was used as another stimulus in parallel. TLR9 mRNA was constitutively expressed at a similar level in purified equine neutrophils across different ages from birth to adulthood, and expression was not affected by either CpG or R. equi. Purified foal neutrophils were directly sensitive to CpG stimulation, reflected by enhanced reactive oxygen species generation following fMLP stimulation, and by expressing significantly (P<0.05) greater mRNA of IFN-gamma, IL-8, IL-12p35, and significantly (P<0.05) decreased TNF-alpha mRNA. In comparison, purified foal neutrophils stimulated by R. equi showed significantly (P<0.05) increased mRNA production of IL-6, IL-8, IL-23p19, and TNF-alpha. Neutrophils co-cultured with other leukocytes expressed a distinct profile of cytokine mRNA than purified neutrophils in response to CpG stimulation, whereas the profile was very similar following R. equi stimulation irrespective of neutrophil purity. When co-cultured with other leukocytes, foal neutrophils were significantly (P<0.05) activated at birth by B-class CpGs and produced IL-6, IL-8, IL-12p40, and IL-23p19 at similar magnitudes to those at 2 months of age. In foal neutrophils at birth, R. equi significantly (P<0.05) induced all cytokines stimulated by CpGs (except IL-12p40), as well as TNF-alpha. Our results indicate that foal neutrophils were sensitive to CpG or R. equi activation as early as at birth, and that B-class CpGs enhanced foal neutrophil functions in vitro.

Neutrophil function of neonatal foals is enhanced in vitro by CpG oligodeoxynucleotide stimulation

Rhodococcus equi is an intracellular bacterium that causes pneumonia in foals and immunocompromised adult horses. Evidence exists that foals become infected with R. equi early in life, a period when innate immune responses are critically important for protection against infection. Neutrophils are innate immune cells that play a key role in defense against this bacterium. Enhancing neutrophil function during early life could thus help to protect foals against R. equi infection. The objective of our study was to determine whether in vitro incubation with the TLR9 agonist CpG 2142 would enhance degranulation and gene expression of cytokines and Toll-like receptor 9 (TLR9) by neutrophils collected from foals at 2, 14, and 56 days of life, and to determine whether these stimulated responses varied among ages. Neutrophil degranulation was enhanced at all ages by in vitro stimulation with either CpG alone, R. equi alone, or in combination with either R. equi or N-formyl-methionyl-leucyl-phenylalanine (fMLP) (P<0.05), but not by in vitro stimulation with fMLP alone. There were no significant differences among ages in CpG-induced cytokine expression, except for IL-12p40, which was induced more at 56 days of age than on days 2 or 14. Collapsing data across ages, CpG 2142 significantly (P<0.05) increased IL-6 and IL-17 mRNA expression. We concluded that in vitro stimulation of foal neutrophils with CpG enhances their function by promoting degranulation and inducing mRNA expression of IL-6 and IL-17, regardless of age.

Gene expression of innate Th1-, Th2-, and Th17-type cytokines during early life of neonatal foals in response to Rhodococcus equi

Focusing on the first 3 weeks of life, this study examined the mRNA transcript development of different Th-type cytokines in foals in response to Rhodococcus equi infection in vitro. Results demonstrated the significant up-regulation in expression of Th1-, Th2-, and Th17-type cytokines (IFN-γ, IL-4, IL-6, IL-8, IL-12p35, IL-12p40, IL-17, IL-23p19, and TNF-α) in R. equi infection of bronchial alveolar lavage (BAL) cells of 10-day-old foals. Consequently, signature cytokines of 3 Th cell types, IFN-γ (Th1), IL-4 (Th2), and IL-17 (Th17), were used to compare temporal response patterns of circulating peripheral blood mononuclear cells (PBMCs) to stimulation with R. equi. Foals responded to R. equi stimulation by producing similar amounts of IFN-γ mRNA transcripts from birth through 3 weeks of age, suggesting an absence of age-related impairment in Th1-type cytokine response to R. equi during the first 3 weeks of life. It remains debatable whether this Th1 response to R. equi in foals≤3 weeks of age is generally immature relative to older foals or adult horses. IL-4 expression by R. equi-stimulated PBMCs was significantly decreased at birth, and IL-17 expression was relatively reduced during the first week of life. Among all cytokines studied, IL-17 mRNA transcripts were induced with the highest magnitude of fold-change both in BAL cells and in PBMCs. Under the conditions studied, in vivo administration of a CpG failed to modulate the Th1-, Th2-, and Th17-type cytokine expression patterns in PBMCs.

Antimicrobial activity of gallium maltolate against Staphylococcus aureus and methicillin-resistant S. aureus and Staphylococcus pseudintermedius: An in vitro study

Gallium is a trivalent semi-metallic element that has shown antimicrobial activity against several important human pathogens. This antimicrobial activity is likely related to its substitution in important iron-dependent pathways of bacteria. The genus Staphylococcus, which includes human and animal pathogens that cause significant morbidity and mortality, requires iron for growth and colonization. In this study, gallium maltolate, at various concentrations between 50 and 200μM, inhibited the in vitro growth of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) at time-points between 8 and 36h after inoculation. The inhibitory activity of gallium maltolate against clinical isolates of MRSA and methicillin-resistant Staphylococcus pseudintermedius (MRSP) from a veterinary teaching hospital was determined.

Effects of Administration of Live or Inactivated Virulent Rhodococccus equi and Age on the Fecal Microbiome of Neonatal Foals

Background Rhodococcus equi is an important pathogen of foals. Enteral administration of live, virulent R. equi during early life has been documented to protect against subsequent intrabronchial challenge with R. equi, indicating that enteral mucosal immunization may be protective. Evidence exists that mucosal immune responses develop against both live and inactivated micro-organisms. The extent to which live or inactivated R. equi might alter the intestinal microbiome of foals is unknown. This is an important question because the intestinal microbiome of neonates of other species is known to change over time and to influence host development. To our knowledge, changes in the intestinal microbiome of foals during early life have not been reported. Thus, the purpose of this study was to determine whether age (during the first month of life) or administration of either live virulent R. equi (at a dose reported to protect foals against subsequent intrabronchial challenge, viz., 1×1010 colony forming units [CFU]) or inactivated virulent R. equi (at higher doses, viz., 2×1010 and 1×1011 [CFU]) altered the fecal microbiome of foals. Methodology/Principal Findings Fecal swab samples from 42 healthy foals after vaccination with low-dose inactivated R. equi (n = 9), high-dose inactivated R. equi (n = 10), live R. equi (n = 6), control with cholera toxin B (CTB, n = 9), and control without CTB (n = 8) were evaluated by 454-pyrosequencing of the 16S rRNA gene and by qPCR. No impact of treatment was observed among vaccinated foals; however, marked and significant differences in microbial communities and diversity were observed between foals at 30 days of age relative to 2 days of age. Conclusions The results suggest age-related changes in the fecal microbial population of healthy foals do occur, however, mucosal vaccination does not result in major changes of the fecal microbiome in foals.

Intramuscular administration of a synthetic CpG-oligodeoxynucleotide modulates functional responses of neutrophils of neonatal foals.

Neutrophils play an important role in protecting against infection. Foals have age-dependent deficiencies in neutrophil function that may contribute to their predisposition to infection. Thus, we investigated the ability of a CpG-ODN formulated with Emulsigen to modulate functional responses of neutrophils in neonatal foals. Eighteen foals were randomly assigned to receive either a CpG-ODN with Emulsigen (N = 9) or saline intramuscularly at ages 1 and 7 days. At ages 1, 3, 9, 14, and 28, blood was collected and neutrophils were isolated from each foal. Neutrophils were assessed for basal and Rhodococcus equi-stimulated mRNA expression of the cytokines interferon-γ (IFN-γ), interleukin (IL)-4, IL-6, and IL-8 using real-time PCR, degranulation by quantifying the amount of β-D glucuronidase activity, and reactive oxygen species (ROS) generation using flow cytometry. In vivo administration of the CpG-ODN formulation on days 1 and 7 resulted in significantly (P<0.05) increased IFN-γ mRNA expression by foal neutrophils on days 3, 9, and 14. Degranulation was significantly (P<0.05) lower for foals in the CpG-ODN-treated group than the control group at days 3 and 14, but not at other days. No effect of treatment on ROS generation was detected. These results indicate that CpG-ODN administration to foals might improve innate and adaptive immune responses that could protect foals against infectious diseases and possibly improve responses to vaccination.

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.

Oral Administration of Electron-Beam Inactivated Rhodococcus equi Failed to Protect Foals against Intrabronchial Infection with Live, Virulent R. equi

There is currently no licensed vaccine that protects foals against Rhodococcus equi–induced pneumonia. Oral administration of live, virulent R. equi to neonatal foals has been demonstrated to protect against subsequent intrabronchial challenge with virulent R. equi. Electron beam (eBeam)-inactivated R. equi are structurally intact and have been demonstrated to be immunogenic when administered orally to neonatal foals. Thus, we investigated whether eBeam inactivated R. equi could protect foals against developing pneumonia after experimental infection with live, virulent R. equi. Foals (n = 8) were vaccinated by gavaging with eBeam-inactivated R. equi at ages 2, 7, and 14 days, or gavaged with equal volume of saline solution (n = 4), and subsequently infected intrabronchially with live, virulent R. equi at age 21 days. The proportion of vaccinated foals that developed pneumonia following challenge was similar among the vaccinated (7/8; 88%) and unvaccinated foals (3/4; 75%). This vaccination regimen did not appear to be strongly immunogenic in foals. Alternative dosing regimens or routes of administration need further investigation and may prove to be immunogenic and protective.

Antibody to Poly-N-acetyl glucosamine provides protection against intracellular pathogens: Mechanism of action and validation in horse foals challenged with Rhodococcus equi

Immune correlates of protection against intracellular bacterial pathogens are largely thought to be cell-mediated, although a reasonable amount of data supports a role for antibody-mediated protection. To define a role for antibody-mediated immunity against an intracellular pathogen, Rhodococcus equi, that causes granulomatous pneumonia in horse foals, we devised and tested an experimental system relying solely on antibody-mediated protection against this host-specific etiologic agent. Immunity was induced by vaccinating pregnant mares 6 and 3 weeks prior to predicted parturition with a conjugate vaccine targeting the highly conserved microbial surface polysaccharide, poly-N-acetyl glucosamine (PNAG). We ascertained antibody was transferred to foals via colostrum, the only means for foals to acquire maternal antibody. Horses lack transplacental antibody transfer. Next, a randomized, controlled, blinded challenge was conducted by inoculating at ~4 weeks of age ~106 cfu of R. equi via intrabronchial challenge. Eleven of 12 (91%) foals born to immune mares did not develop clinical R. equi pneumonia, whereas 6 of 7 (86%) foals born to unvaccinated controls developed pneumonia (P = 0.0017). In a confirmatory passive immunization study, infusion of PNAG-hyperimmune plasma protected 100% of 5 foals against R. equi pneumonia whereas all 4 recipients of normal horse plasma developed clinical disease (P = 0.0079). Antibodies to PNAG mediated killing of extracellular and intracellular R. equi and other intracellular pathogens. Killing of intracellular organisms depended on antibody recognition of surface expression of PNAG on infected cells, along with complement deposition and PMN-assisted lysis of infected macrophages. Peripheral blood mononuclear cells from immune and protected foals released higher levels of interferon-γ in response to PNAG compared to controls, indicating vaccination also induced an antibody-dependent cellular release of this critical immune cytokine. Overall, antibody-mediated opsonic killing and interferon-γ release in response to PNAG may protect against diseases caused by intracellular bacterial pathogens.

Chloroquine inhibits Rhodococcus equi replication in murine and foal alveolar macrophages by iron-starvation.

Rhodococcus equi preferentially infects macrophages causing pyogranulomatous pneumonia in young foals. Both the vapA and rhbC genes are up-regulated in an iron (Fe)-deprived environment, such as that found within macrophages. Chloroquine (CQ) is a drug widely used against malaria that suppresses the intracellular availability of Fe in eukaryotic cells. The main objective of this study was to evaluate the ability of CQ to inhibit replication of virulent R. equi within murine (J774A.1) and foal alveolar macrophages (AMs) and to verify whether the mechanism of inhibition could be Fe-deprivation-dependent. CQ effect on R. equi extracellular survival and toxicity to J774A.1 were evaluated. R. equi survival within J774A.1 and foal AMs was evaluated under CQ (10 and 20μM), bovine saturated transferrin (bHTF), and bovine unsaturated transferrin (bATF) exposure. To explore the action mechanism of CQ, the superoxide anion production, the lysozyme activity, as well as the relative mRNA expression of vapA and rhbC were examined. CQ at≤20μM had no effect on R. equi extracellular multiplication and J774A.1 viability. Exposure to CQ significantly and markedly reduced survival of R. equi within J774A.1 and foal AMs. Treatment with bHTF did not reverse CQ effect on R. equi. Exposure to CQ did not affected superoxide anion production or lysozyme activity, however vapA and rhbC expression was significantly increased. Our results reinforce the hypothesis that intracellular availability of Fe is required for R. equi survival, and our initial hypothesis that CQ can limit replication of R. equi in J774A.1 and foal AMs, most likely by Fe starvation.