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., 1×10(10) colony forming units [CFU]) or inactivated virulent R. equi (at higher doses, viz., 2×10(10) and 1×10(11) [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.The open access fee for this work was funded through the Texas A&M University Open Access to Knowledge (OAK) Fund

Rarefaction analysis of 16 S rRNA gene sequences obtained from fecal swabs from foals.

<p>Lines represent the average of each vaccination group at all ages (panel A) or at 30 days only (panel B), while the error bars represent the standard deviations. The analysis was performed on a randomly selected subset of 1,300 sequences per sample and included samples from 42 foals. Note that both the greatest and least number of species observed occurred among foals that received no enteral bacteria (live or inactivated), indicating an absence of evidence of treatment effect. Control  =  control plus CTB group; Control_no_CTO  =  control without CTB group; High  =  high-dose inactivated <i>R. equi</i> group; Live  =  live <i>R. equi</i> group; Low  =  low-dose inactivated <i>R. equi</i> group.</p

Median and range proportion of foals with sequences detected in the fecal DNA of rectal swab samples (Phylum, class, order, and family).

<p>Fecal swab samples collected from 37 Quarter Horse foals on days 2 and 30 of life. *P values represent the results of McNemar’s test for paired dichotomous data, adjusted by the method of Hochberg. NP  =  Not Performed.</p

Summary of alpha diversity measures.

<p>Summary of alpha diversity measures.</p

Principal coordinates analysis (PCoA) of unweighted UniFrac distances of 16 S rRNA genes.

<p>Analysis for 42 foals in groups control with CTB (red square), control without CTB (yellow triangle), low-dose inactivated <i>R. equi</i> (dark blue triangle), high-dose inactivated <i>R. equi</i> 2 (green dot), and live <i>R. equi</i> (light blue triangle) at 30 days of age only. Differences among groups were not significant (ANOSIM, P = 0.449). The 3 panels represent the comparison of the first 2 principal components (A), the second and third principal components (B), and the first and third principal components (C).</p

Results of qPCR analysis.

<p>Median (range) of log DNA. *P value for Wilcoxon rank-sum test comparing differences between ages day 30 and day 2, adjusted by the method of Hochberg.</p

Median and range percentages of sequences represented in the fecal DNA of rectal swab samples from foals (Phylum, class, order, and family).

<p>Fecal swab samples were collected from 37 Quarter Horse foals on days 2 and 30 of life. *P values represent the results of Wilcoxon sign-rank tests for paired differences, adjusted by the method of Hochberg. NP  =  Not Performed.</p

Mean (and 95% confidence intervals) for mRNA copy numbers estimated by mixed-effects modeling for IL-4, IL-6, and IL-8.

<p>Data represent back-transformed results of log₁₀-transformed data. Values in columns with the same letter indicate absence of statistical significance between groups for a given age. Values in rows with differing superscripted numbers indicate significant (P<0.05) differences among ages within group.</p><p>Mean (and 95% confidence intervals) for mRNA copy numbers estimated by mixed-effects modeling for IL-4, IL-6, and IL-8.</p

Mean (and 95% confidence intervals) for neutrophil parameters determined using flow cytometry (please see text for details) and estimated by mixed-effects modeling.

<p>Values in columns with the same letter indicate absence of statistical significance between groups for a given age. Values in rows with differing superscripted numbers indicate significant (P<0.05) differences among ages within group.</p><p>Mean (and 95% confidence intervals) for neutrophil parameters determined using flow cytometry (please see text for details) and estimated by mixed-effects modeling.</p