Prevalence of Bacteria Genera in Rhesus macaques.

a<p>Average of sequences.</p>b<p>Percent of macaques with >1% of sequences corresponding to this genus.</p

In Captive Rhesus Macaques, Cervicovaginal Inflammation Is Common but Not Associated with the Stable Polymicrobial Microbiome

<div><p>Vaginal inoculation of rhesus macaques (RM) with simian immunodeficiency virus (SIV) has been used to study the biology of HIV transmission. Although the results of vaginal SIV transmission experiments could be affected by vaginal inflammation, studies to date have been conducted without regard to levels of pre-existing genital inflammation present in RM. We collected cevicovaginal secretions (CVS) from 33–36 RM during the mid menstrual cycle (day 10–20) at 2 time points approximately 8 months apart and characterized the mRNA and protein levels of inflammatory cytokines, chemokines and interferon-stimulated genes. There was extreme variability in the levels of inflammatory mediators (IFN-α, IFN-γ, IL-6, TNF, IL-1b, IP-10, MIG, IL-12 and IL-17). In most animals, the mRNA levels of the inflammatory mediators were similar in the 2 CVS samples collected 8 months apart, suggesting that genital inflammation is stable in a subset of captive female RM. At both time points the cervicovaginal microbiota had low levels of <em>Lactobacillus</em> and was relatively diverse with an average of 13 genera in the samples from the first time point (median 13, range 7–21) and an average of 11.5 genera in the samples from the second time point (median 11, range 5–20). Many of the macaques had similar microbiota in the samples collected 8 months apart. However, we found no correlation between specific bacterial genera and the mRNA or protein levels of the inflammatory mediators in the genital tract of RM in this study. It seems likely that results of published vaginal SIV transmission experiments in RM have been influenced by pre-existing inflammation in the animals used for the experiments.</p> </div

Concentration of all mRNAs (relative to GAPDH) in vaginal secretions collected between menstrual cycle days 10–20 from 36 RM at Time point 1 (March 2011) and from 30–35 RM at Time point 2 (November 2011).

<p>The samples collected at Time point 2 are denoted by the notation “−2”. All vaginal secretions were collected between menstrual cycle days 10–20. Note that there was not enough CVS sample at Time point 2 to assess all mRNA targets that were tested at Time point 1. Grey bars denote median and interquartile range of the values.</p

Genera of macaque lower genital tract bacteria.

<p>The genital microbiota in 21 macaques was identified at two times (approximately 8 months apart). Each group of two bars represents the relative proportions of 16S sequences indentifying bacterial genera in one macaque at the two different time points. Only the 15 most predominant genera are displayed for clarity.</p

Concentration of A) cytokine and B) chemokine proteins measured in cervicovaginal secretions of RM.

<p>All samples were collected between menstrual cycle days 10–20 from 19–22 RM at Time point 2. Bars denote median and interquartile range. Note that if an assay produced a concentration of an analyte below the minimum quantifiable level, a value of zero was assigned and no data points for that sample appears in the graphs.</p

Comparison of relative concentration of for pro-inflammatory cytokine/chemokine mRNAs in vaginal secretions of RM in samples are similar.

<p>A) IL-6, B) TNF, C) MIP-1a, D) MIP-1b, E) IFNa, F) MIG. Time 1 indicates the CVS samples were collected in March 2011. Time 2 indicates that the CVS samples were collected in November 2011. All vaginal secretions were collected between menstrual cycle days 10–20.</p

Network of statistical correlations between mRNA levels of immune mediators.

<p>After unbiased analysis of potential associations between the levels of every mRNA levels measured using a Spearman’s correlation function there was a limited network of strong (>0.7) correlations between mRNA levels of A) 3 cytokine/chemokines at Time point 1; B) 3 cytokines/chemokines; and 2 Interferon-stimulated genes at Time point 2. C) networks of strong correlations that existed at both Time 1 and Time 2. Blue circles indicate host gene mRNA levels. The lines indicate a positive correlation between the parameters in the circles and the width of the line is proportional to the strength of the correlation.</p

Network of statistical correlations between microbiota.

<p>A. Strong (>0.7) correlations between Microbiota at time point 1. B. Intersection of strong correlations that existed at both time 1 and time 2. Pink circles bacterial DNA levels. The blue lines indicate a positive correlation between the parameters in the circles and the width of the line is proportional to the strength of the correlation.</p

Network of statistical correlations between inflammatory mediators and microbiota.

<p>After unbiased analysis of potential associations between host gene mRNA levels and bacterial DNA levels using a Spearman’s correlation function there was a limited network of strong (>0.7) correlations between chemokines/cytokines and microbiota A) at time point 1; B) at time point 2. C) Intersection of strong correlations that existed at both Time 1 and Time 2. Blue circles, host gene mRNA levels, green circles host protein levels, pink circles bacterial DNA levels. The blue lines indicate a positive correlation between the parameters in the circles and the width of the line is proportional to the strength of the correlation. The red lines indicate a negative correlation between the parameters in the circles and the width of the line is proportional to the strength of the correlation.</p

Influenza virus replication in the lower respiratory tract after A/Memphis/7/01 inoculation.

<p>Mean infectious virus titer in tracheal secretions (TCID₅₀/ml) of nine inoculated rhesus macaques.</p