Rhodococcus equi is a common cause of severe pneumonia in foals, and is an opportunistic pathogen in immunocompromised humans. In combination, erythromycin and rifampin are the most commonly used antimicrobials in treating R. equi in foals. To provide reliable treatment, it is imperative to determine the mean inhibitory concentrations (MICs) of other antimicrobial agents in the event that certain strains of R. equi develop resistance to the current treatment. Several strains of R. equi have developed resistance to various antibiotics. In this study, R. equi strain 288 was completely resistant to rifampin with a MIC > 256ug/ml. The MICs of ethambutol, clarithromycin, azithromycin, isoniazide, ethionamide, rifampin, erythromycin, and linezolid of ninety-five R. equi isolates were also determined in this study. These isolates were obtained from the lungs and transtracheal washes of foals. In addition to these strains, three National Committee for Laboratory Clinical Standards (NCCLS) quality control strains were also tested: R. equi ATCC 6939, R. equi ATCC 33701, and S. pneumoniae 49619. Each drug was tested in triplicate and the MIC 50’s and MIC 90’s were determined for each drug. Ethambutol, isoniazide, and ethionamide were completely ineffective against R. equi. with MICs > 250ug/ml. Rhodococcus equi strains were more susceptible to clarithromycin (MIC 90 = 0.23 ug/ml) than to azithromycin (MIC 90 = 2.33 ug/ml), rifampicin (MIC 90 = 0.67ug/ml), erythromycin (MIC 90 = 1.2ug/ml), and linezolid (MIC 90 = 4ug/ml).

Blood was obtained from 5 adult horses and 16 juvenile horses (foals) at the time of birth and subsequently at 2-, 4-, and 8-weeks of age. Neutrophils from adult horses were purified and incubated for 2 h and 4 h with media, avirulent R. equi, virulent R. equi, or recombinant-human granulocyte-macrophage colony stimulating factor (rhGM-CSF). Neutrophils from foals were purified and incubated for 2 h and 4 h with media or virulent R. equi. Total RNA was extracted from both adult and foal neutrophils immediately after purification to measure baseline expression levels (0 h), and immediately after each of the prescribed incubation times. For each sample, 1 µg of total RNA was reverse-transcribed and analyzed for differential gene expression using real-time PCR. After 2 h and 4 h incubation with virulent or avirulent R. equi, neutrophils from adult horses expressed significantly (P< 0.05) greater TNFα, IL-12p40, IL-6, IL-8, and IL-23p19 mRNA relative to expression by unstimulated neutrophils, but not IFNγ or IL-12p35 mRNA. Furthermore, virulent R. equi induced significantly greater IL-23p19 mRNA expression than avirulent R. equi. Stimulation with rhGM-CSF of adult equine neutrophils failed to induce significant changes in cytokine expression. In foal neutrophils, stimulation with virulent R. equi induced significantly greater expression of IFNγ, TNFα, IL-6, IL-8, IL-12p40, and IL-12p35 mRNA relative to expression by unstimulated neutrophils. Furthermore, there were significant effects of age on expression of IL-6, IL-8 and IL-12p40 mRNA. Neutrophil mRNA expression of IL-6 and IL-8 in newborn foals was significantly greater than expression at 2-, 4-, and 8-weeks of age. There was no significant difference between unstimulated and R. equi-stimulated neutrophils from newborn and 2-week-old foals in expression of IL-12p40; however, expression of IL-12p40 by R. equi-stimulated neutrophils from 4- and 8-week-old foals was significantly greater than expression by unstimulated neutrophils. These results demonstrate that R. equi-stimulated neutrophils are a source of many pro-inflammatory cytokines, and that the magnitude of this expression with respect to IL-6, IL-8, and IL-12p40 mRNA expression was influenced by age. Collectively, the data presented indicate a non-phagocytic role for neutrophils that may influence the type of adaptive immune response to R. equi.

Blood was obtained from 5 adult horses and 16 juvenile horses (foals) at the time of birth and subsequently at 2-, 4-, and 8-weeks of age. Neutrophils from adult horses were purified and incubated for 2 h and 4 h with media, avirulent R. equi, virulent R. equi, or recombinant-human granulocyte-macrophage colony stimulating factor (rhGM-CSF). Neutrophils from foals were purified and incubated for 2 h and 4 h with media or virulent R. equi. Total RNA was extracted from both adult and foal neutrophils immediately after purification to measure baseline expression levels (0 h), and immediately after each of the prescribed incubation times. For each sample, 1 µg of total RNA was reverse-transcribed and analyzed for differential gene expression using real-time PCR. After 2 h and 4 h incubation with virulent or avirulent R. equi, neutrophils from adult horses expressed significantly (P< 0.05) greater TNFα, IL-12p40, IL-6, IL-8, and IL-23p19 mRNA relative to expression by unstimulated neutrophils, but not IFNγ or IL-12p35 mRNA. Furthermore, virulent R. equi induced significantly greater IL-23p19 mRNA expression than avirulent R. equi. Stimulation with rhGM-CSF of adult equine neutrophils failed to induce significant changes in cytokine expression. In foal neutrophils, stimulation with virulent R. equi induced significantly greater expression of IFNγ, TNFα, IL-6, IL-8, IL-12p40, and IL-12p35 mRNA relative to expression by unstimulated neutrophils. Furthermore, there were significant effects of age on expression of IL-6, IL-8 and IL-12p40 mRNA. Neutrophil mRNA expression of IL-6 and IL-8 in newborn foals was significantly greater than expression at 2-, 4-, and 8-weeks of age. There was no significant difference between unstimulated and R. equi-stimulated neutrophils from newborn and 2-week-old foals in expression of IL-12p40; however, expression of IL-12p40 by R. equi-stimulated neutrophils from 4- and 8-week-old foals was significantly greater than expression by unstimulated neutrophils. These results demonstrate that R. equi-stimulated neutrophils are a source of many pro-inflammatory cytokines, and that the magnitude of this expression with respect to IL-6, IL-8, and IL-12p40 mRNA expression was influenced by age. Collectively, the data presented indicate a non-phagocytic role for neutrophils that may influence the type of adaptive immune response to R. equi.

Rhodococcus equi is a common cause of severe pneumonia in foals, and is an opportunistic pathogen in immunocompromised humans. In combination, erythromycin and rifampin are the most commonly used antimicrobials in treating R. equi in foals. To provide reliable treatment, it is imperative to determine the mean inhibitory concentrations (MICs) of other antimicrobial agents in the event that certain strains of R. equi develop resistance to the current treatment. Several strains of R. equi have developed resistance to various antibiotics. In this study, R. equi strain 288 was completely resistant to rifampin with a MIC > 256ug/ml. The MICs of ethambutol, clarithromycin, azithromycin, isoniazide, ethionamide, rifampin, erythromycin, and linezolid of ninety-five R. equi isolates were also determined in this study. These isolates were obtained from the lungs and transtracheal washes of foals. In addition to these strains, three National Committee for Laboratory Clinical Standards (NCCLS) quality control strains were also tested: R. equi ATCC 6939, R. equi ATCC 33701, and S. pneumoniae 49619. Each drug was tested in triplicate and the MIC 50’s and MIC 90’s were determined for each drug. Ethambutol, isoniazide, and ethionamide were completely ineffective against R. equi. with MICs > 250ug/ml. Rhodococcus equi strains were more susceptible to clarithromycin (MIC 90 = 0.23 ug/ml) than to azithromycin (MIC 90 = 2.33 ug/ml), rifampicin (MIC 90 = 0.67ug/ml), erythromycin (MIC 90 = 1.2ug/ml), and linezolid (MIC 90 = 4ug/ml).