Persistent anthrax as a major driver of wildlife mortality in a tropical rainforest

Anthrax is a globally important animal disease and zoonosis. Despite this, our current knowledge of anthrax ecology is largely limited to arid ecosystems, where outbreaks are most commonly reported. Here we show that the dynamics of an anthrax-causing agent, Bacillus cereus biovar anthracis, in a tropical rainforest have severe consequences for local wildlife communities. Using data and samples collected over three decades, we show that rainforest anthrax is a persistent and widespread cause of death for a broad range of mammalian hosts. We predict that this pathogen will accelerate the decline and possibly result in the extirpation of local chimpanzee (Pan troglodytes verus) populations. We present the epidemiology of a cryptic pathogen and show that its presence has important implications for conservation

Persistent anthrax as a major driver of wildlife mortality in a tropical rainforest

Anthrax is a globally important animal disease and zoonosis. Despite this, our current knowledge of anthrax ecology is largely limited to arid ecosystems, where outbreaks are most commonly reported. Here we show that the dynamics of an anthrax-causing agent, Bacillus cereus biovar anthracis, in a tropical rainforest have severe consequences for local wildlife communities. Using data and samples collected over three decades, we show that rainforest anthrax is a persistent and widespread cause of death for a broad range of mammalian hosts. We predict that this pathogen will accelerate the decline and possibly result in the extirpation of local chimpanzee (Pan troglodytes verus) populations. We present the epidemiology of a cryptic pathogen and show that its presence has important implications for conservation

CD4⁺ T Cells Are as Protective as CD8⁺ T Cells against <i>Rickettsia typhi</i> Infection by Activating Macrophage Bactericidal Activity

<div><p><i>Rickettsia typhi</i> is an intracellular bacterium that causes endemic typhus, a febrile disease that can be fatal due to complications including pneumonia, hepatitis and meningoencephalitis, the latter being a regular outcome in T and B cell-deficient C57BL/6 RAG1^-/- mice upon <i>Rickettsia typhi</i> infection. Here, we show that CD4⁺ T_H1 cells that are generated in C57BL/6 mice upon <i>R</i>. <i>typhi</i> infection are as protective as cytotoxic CD8⁺ T cells. CD4⁺- as well as CD8⁺-deficient C57BL/6 survived the infection without showing symptoms of disease at any point in time. Moreover, adoptively transferred CD8⁺ and CD4⁺ immune T cells entered the CNS of C57BL/6 RAG1^-/- mice with advanced infection and both eradicated the bacteria. However, immune CD4⁺ T cells protected only approximately 60% of the animals from death. They induced the expression of iNOS in infiltrating macrophages as well as in resident microglia in the CNS which can contribute to bacterial killing but also accelerate pathology. <i>In vitro</i> immune CD4⁺ T cells inhibited bacterial growth in infected macrophages which was in part mediated by the release of IFNγ. Collectively, our data demonstrate that CD4⁺ T cells are as protective as CD8⁺ T cells against <i>R</i>. <i>typhi</i>, provided that CD4⁺ T_H1 effector cells are present in time to support bactericidal activity of phagocytes via the release of IFNγ and other factors. With regard to vaccination against TG <i>Rickettsiae</i>, our findings suggest that the induction of CD4⁺ T_H1 effector cells is sufficient for protection.</p></div

Immune CD4⁺ T cells induce NO release by <i>R</i>. <i>typhi</i>-infected macrophages <i>in vitro</i> and inhibit bacterial growth via IFNγ and TNFα.

<p>1×10⁶ bone-marrow-derived BALB/c macrophages were infected with 5 copies of <i>R</i>. <i>typhi</i> per cell one day prior to the addition of 2×10⁶ purified CD4⁺ T cells from either naïve or immune BALB/c mice (day 7 post infection). IFNγ and TNFα were neutralized by the addition of 10 μg/ml anti-IFNγ and/or anti-TNFα as indicated on the x-axis. Cytokines were quantified in the supernatants 72h after T cell addition by LEGENDplex assay. IFNγ (left, y-axis), TNFα (middle, y-axis), IL-22 (right, y-axis) and IL-2 (below, left) are shown. Other cytokines were not detectable (<b>A</b>). In addition, NO was detected 72h after T cell addition (<b>B</b>). Bacterial content in the cultures (y-axis) was assessed by <i>prsA</i>-specific qPCR 72h after T cell addition (<b>C</b>). 1×10⁶ bone-marrow-derived BALB/c macrophages were treated with recombinant IFNγ (1 U/ml) or TNFα (400 U/ml). NO was quantified in the cell culture supernatants after 72h (<b>D</b>). 1×10⁶ bone-marrow-derived BALB/c macrophages were infected with 5 copies of <i>R</i>. <i>typhi</i> per cell one day prior to the addition of recombinant IFNγ (1 U/ml) or TNFα (400 U/ml). The cytokines were neutralized by simultaneous addition of either anti-TNFα or anti-IFNγ (10 μg/ml each) as indicated on the x-axis. Bacterial content in the cultures (y-axis) was assessed by <i>prsA</i>-specific qPCR 72h after cytokine addition (<b>E</b>). Graphs show the mean±SEM of combined results from 2 independent experiments (n = 4 T cells from each group of mice (A-C) and n = 2 for the treatment with recombinant cytokines (D-E)). Statistical analysis was performed by One-way ANOVA (Kruskal-Wallis test followed by Dunn´s post test). Asterisks indicate significant differences (*<i>p</i><0.05, **<i>p</i><0.01).</p

Detection of infiltrating T cells, IBA1⁺ cells and iNOS expression in sagittal sections of the brains from <i>R</i>. <i>typhi</i>-infected C57BL/6 RAG1^-/- control mice, CD4⁺ and CD8⁺ recipients (day 7 post transfer).

<p>Sagittal sections were further stained for CD3 to detect infiltrating T cells, for IBA1 that is expressed on infiltrating MΦ as well as by microglia and for iNOS. Sections from the brains of CD4⁺ T cell recipients that were not infected were used as additional control (<b>A</b>). Representative stainings of the brain from a <i>R</i>. <i>typhi</i>-infected C57BL/6 RAG1^-/- control mouse (<b>B</b>), a <i>R</i>. <i>typhi</i>-infected mouse that received CD4⁺ T cells (<b>C</b>) and a CD8⁺ T cell recipient (<b>D</b>) are shown.</p

Bacterial content in the organs of surviving CD8⁺Perforin^-/-, CD8⁺IFNγ^-/- and CD4⁺IFNγ^-/- T cell recipients.

<p>Bacterial content in the organs of surviving CD8⁺Perforin^-/-, CD8⁺IFNγ^-/- and CD4⁺IFNγ^-/- T cell recipients.</p

Immune CD4⁺ T cells and IFNγ activate bmMΦ for bacterial killing <i>in vitro</i>.

<p>2×10⁶ bmMΦ were infected with 5 copies of <i>R</i>. <i>typhi</i> per cell. Medium was exchanged 3h post infection. 24h after infection 1.5×10⁶ purified CD4⁺ T cells from either PBS-treated control (white bars) or <i>R</i>. <i>typhi</i>-infected C57BL/6 wild-type mice (day 7 post infection; black bars) were added. IFNγ was neutralized by simultaneous addition of anti- IFNγ (1 μg/ml; striped bar). Cytokines and NO were quantified in the cell culture supernatant (y-axis) 96h post bmMΦ infection (48h after addition of T cells or IFNγ) and bacterial growth was assessed by qPCR 96h post bmMΦ infection. Graph shows combined results from two independent experiments for each of which T cells from 6 individual control or <i>R</i>. <i>typhi</i>-infected mice were used (<b>A</b>). 2×10⁶ bmMΦ were infected with 5 copies of <i>R</i>. <i>typhi</i> per cell. Medium was exchanged 3h post infection and either replaced by unconditioned medium (gray bar), medium containing recombinant IFNγ (10 U/ml; black bar) or the same amount of recombinant IFNγ that was pre-incubated with neutralizing anti-IFNγ (1 μg/ml; gray striped bar). Bars show combined results from two independent experiments for each of which MΦ from 3 individual C57BL/6 mice were used (<b>B</b>). Statistical analysis was performed with Kruskal-Wallis test followed by Dunn´s post test. Asterisks indicate statistically significant differences (*<i>p</i><0.05, ***<i>p</i><0.001).</p

Enhanced protection by CD4⁺IFNγ^-/- T cells in the absence of TNFα.

<p>1×10⁶ bone-marrow-derived BALB/c macrophages were infected with 5 copies of <i>R</i>. <i>typhi</i> per cell one day prior to the addition of 2×10⁶ purified CD4⁺ T cells from either naïve or immune BALB/c IFNγ^-/- mice (day 7 post infection). TNFα was neutralized by simultaneous addition of 10 μg/ml anti-TNFα. Bacterial content in the cultures (y-axis) was assessed by <i>prsA</i>-specific qPCR 72h after T cell addition. Graphs show the mean and SEM of combined results from two independent experiments (n = 4 T cells from each group of mice) (<b>A</b>). CB17 SCID mice (n = 7 for each group) were infected with 1×10⁶ sfu <i>R</i>. <i>typhi</i>. 1×10⁶ purified CD4⁺ T cells from BALB/c IFNγ^-/- mice were adoptively transferred one day prior to the infection with <i>R</i>. <i>typhi</i>. Control groups of mice received PBS instead. TNFα was neutralized by intraperitoneal application of 500 μg anti-TNFα every three days beginning on day 3 post infection. Control animals received equal amounts of isotype antibody. The state of health of the mice was monitored by weight change (y-axis, upper left) and a clinical score (y-axis, upper right) and the survival rates (y-axis, below) were assessed. Dotted lines show the data for surviving animals of the isotype- and anti-TNFα-treated groups of CD4⁺IFNγ^-/- recipients. Statistical analysis of survival rates was performed with Log-rank (Mantel-Cox) test. Asterisks indicate significant differences compared to control animals (**<i>p</i><0.01) (<b>B</b>). Serum GPT levels (y-axis) were assessed from all groups of animals at the time of death and in surviving animals at the end of the experiment (day 34). Combined results are shown. Each dot represents a single mouse. Statistical analysis was performed by One-way ANOVA (Kruskal Wallis test followed by Dunn´s post test) (*<i>p</i><0.05) (<b>C</b>). The bacterial content (y-axis) in the organs was quantified by <i>prsA</i>-specific qPCR from all animals that succumbed to the infection at the time of death and from surviving animals at the end of the experiment (day 34) as indicated on the x-axis (<b>D</b>). Statistical analysis for C and D was performed by One-way ANOVA (Kruskal-Wallis test followed by Dunn´s post test). Asterisks indicate statistically significant differences (*<i>p</i><0.05, **<i>p</i><0.01).</p

BALB/c mice generate cytotoxic CD8⁺ cells that are sporadically reactivated.

<p>BALB/c mice were infected with 1×10⁶ sfu <i>R</i>. <i>typhi</i>. Control mice received PBS instead and were used as "day 0" control. Spleen cells were isolated and stained for CD8, KLRG1 and CD11a or restimulated with PMA/Ionomycin for 4h and stained for CD8 and intracellular IFNγ and Granzyme B. The dot plots show example stainings from day 7 post infection. Mice were analyzed for cytokine and Granzyme B expression on day 0, 7 and 15 (n = 6) and day 35 (n = 4). 3–4 mice were analyzed for KLRG1 and CD11a expression. Graphs show the percentage of KLRG1⁺, CD11a⁺, Granzyme B⁺ and IFNγ⁺ T cells among CD8⁺ T cells (y-axis) at indicated days post infection (x-axis). Graphs show combined results from 2 independent experiments. Statistical analysis was performed by One-way ANOVA (Kruskal Wallis test followed by Dunn´s post test). Asterisks indicate significant differences compared to day 0 (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001).</p

MΦ do not react to <i>R</i>. <i>typhi</i> in a classical manner and are incapable to kill the bacteria <i>in vitro</i>.

<p>bmMΦ were left untreated, stimulated with LPS (500 ng/ml) as a control or infected with indicated amounts of <i>R</i>. <i>typhi</i> copies, stimulated with LPS (500 ng/ml) or left untreated (-) as indicated on the x-axis. Bacterial content, the expression of CD80, MHCI and MHCII was analyzed after 24h and 48h by flow cytometry. In addition, <i>R</i>. <i>typhi</i> was stained for immunofluorescence microscopy 48h after infection (insertion). Graphs show the percentage of <i>R</i>. <i>typhi</i>-positive cells (y-axis) and the mean fluorescence intensity (MFI) minus the mean MFI of untreated cells (ΔMFI, y-axis). The graphs show combined results from the stimulation of bmMΦ derived from four individual C57BL/6 mice. Statistical analysis was performed with Kruskal-Wallis test and Dunn´s post test. Asterisks indicate statistically significant differences compared to untreated cells (*<i>p</i><0.05) (<b>A</b>). The concentration (y-axis) of the indicated cytokines and NO (x-axis) was quantified in the supernatants from untreated (white bars) or LPS-stimulated bmMΦ (gray bars) and from bmMΦ cultures that were infected with 50 <i>R</i>. <i>typhi</i> copies per cell (black bars). Cytokines and NO were determined at 48h post inoculation. Statistical analysis was performed with Kruskal-Wallis test and Dunn´s post test. Asterisks indicate statistically significant differences compared to untreated cells (*<i>p</i><0.05**, <i>p</i><0.01, ***<i>p</i><0.001) (<b>B</b>). bmMΦ were infected with 5 <i>R</i>. <i>typhi</i> copies per cell. Medium was exchanged after 3h and cells were further incubated for 96h. Copy numbers of <i>R</i>. <i>typhi</i> (y-axis) were determined at indicated points in time (x-axis) by qPCR. The graph shows combined results from the stimulation of bmMΦ from four individual C57BL/6 mice. Statistical analysis was performed with Mann-Whitney U test. Asterisks indicate statistically significant differences compared to non-infected control cells (*<i>p</i><0.05) (<b>C</b>).</p