Opsonization but not pretreatment of equine macrophages with hyperimmune plasma nonspecifically enhances phagocytosis and intracellular killing of Rhodococcus equi.

BACKGROUND: Evidence regarding the efficacy of equine hyperimmune plasma to prevent pneumonia in foals caused by Rhodococcus equi is limited and conflicting. HYPOTHESIS: Opsonization with R. equi-specific hyperimmune plasma (HIP) will significantly increase phagocytosis and decrease intracellular replication of R. equi by alveolar macrophages (AMs) compared to normal plasma (NP). ANIMALS: Fifteen adult Quarter Horses were used to collect bronchoalveolar lavage cells. METHODS: In the first experiment, AMs from 9 horses were pretreated (incubated) with either HIP, NP, or media only (control) and then infected with nonopsonized R. equi. In a second experiment, AMs from 6 horses were infected with R. equi either opsonized with HIP or opsonized with NP. For both experiments, AMs were lysed at 0 and 48 hours and the number of viable R. equi quantified by culture were compared among groups using linear mixed-effects modeling with significance set at P < .05. RESULTS: Opsonization with either HIP or NP increased phagocytosis by AMs (P < .0001) and decreased intracellular survival of organisms in AMs (P < .0001). Pretreating AMs with either HIP or NP without opsonizing R. equi had no effects on phagocytosis or intracellular replication. CONCLUSIONS AND CLINICAL IMPORTANCE: Opsonizing R. equi with either NP or HIP decreases intracellular survival of organisms in AMs, but the effect does not appear to be enhanced by using HIP. Mechanisms other than effects on AMs must explain any clinical benefits of using HIP over NP to decrease the incidence of R. equi pneumonia in foals.

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 ~10(6) 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.