<i>Brucella</i> specifically targets and multiplies in cervical lymph nodes during oral infection and causes long-term lymphadenopathy.

<p>C57BL/6 mice were infected by gavage or by the oral route with 10⁹<i>B</i>. <i>melitensis</i> per mouse. At 2, 8, 29 or 50 days post-infection, mice were sacrificed and organs weighed and analyzed for their bacterial loads by plating homogenates on nutrient agar. Data represent mean organ weight or CFU per organ and SEM of the pooled results from two independent experiments with 4 mice per group.</p

<i>Brucella</i> and fluorescent microspheres in the CLN localize in cells positive for CD68 and low or negative for CD11c.

<p><b>(A)</b> C57BL/6 mice were fed by oral gavage with 0.2 μm yellow green fluorescent microspheres. After 3 days, they were sacrificed and CLN processed for immunofluorescence microscopy. <b>(B)</b> Cells with internal beads from experiments as shown in (A) were quantified as to their expression of CD11c and CD68. At least 100 bead-containing cells per experiment were counted. <b>(C)</b> Mice infected by the oral route with 10⁹<i>B</i>. <i>melitensis</i> per mouse were sacrificed at day 8, CLN prepared for immunofluorescence analysis as described above and <b>(D)</b> the number of infected cells positive for either marker was determined. All available cuts from the CLN of one mouse were analyzed. Data shown represents mean and standard deviation of three independent experiments. Bars: 10 μm.</p

<i>B</i>. <i>melitensis</i> oral infection results in an increase of CLN CD11b^high cells expressing either F4/80, Ly6c or both.

<p>CLN from mice orally infected with 10⁹<i>B</i>. <i>melitensis</i> per mouse for 15 days were prepared for flow cytometry. Total CLN cell numbers were analyzed for the respective percentages <b>(A)</b> or absolute numbers <b>(B)</b> of dendritic cells (F4/80^-CD11c^highMHCII^int and F4/80^-CD11c^intMHCII^high), CD11b^high macrophages/monocytes (F4/80^-Ly6c⁺, F4/80⁺Ly6c⁺ and F4/80⁺Ly6c^-) and neutrophils (CD11b^highLy6G⁺). <b>(C)</b> shows a representative contour plot of respective populations from a mock-infected or <i>Brucella</i>-infected mouse on CD19^-Ly6G^-CD11b^high (F4/80 vs. Ly6c) or CD19^-CD11b^low/intF4/80^-NK1.1^- cells (CD11c vs. MHCII). Populations shown in (A) were analyzed for their median fluorescence of <b>(D)</b> CD11b, <b>(E)</b> CD11c and <b>(F)</b> MHCII. Data represent mean and SEM of pooled results from two independent experiments with a total of 8 (mock-infected) and 9 (infected) mice per group. * p ≤ 0.05 as compared to respective mock infected control.</p

Infection by the oral route leads to preferential bacterial colonization of the CLN.

<p>C57BL/6 mice were infected by intraperitoneal injection (10⁶ bacteria/mouse), intragastric by gavage or by the oral route (both at 10⁹ bacteria/mouse). At 8 days post-infection, mice were sacrificed and organs weighed and analyzed for their bacterial loads by plating homogenates on nutrient agar. Data represent mean organ weight or colony forming units (CFU) per organ and SEM of the pooled results from two independent experiments with 5 mice per group. Non-infected organs are not shown due to logarithmic scale. * p ≤ 0.05. CLN—cervical lymph nodes; MLN—mesenteric lymph nodes.</p

Infection with decreasing bacterial numbers by gavage results in increasingly specific bacterial colonization of the CLN.

<p>C57BL/6 mice were infected by gavage with 10⁹, 10⁸ or 10⁷<i>B</i>. <i>melitensis</i> per mouse. At 8 days post-infection, mice were sacrificed and organs weighed and analyzed for their bacterial loads by plating homogenates on nutrient agar. Data represent mean CFU per organ and SEM of the pooled results from two independent experiments with 3 mice per group. Non-infected organs are not shown due to logarithmic scale. * p ≤ 0.05. CLN—cervical lymph nodes; MLN—mesenteric lymph nodes; ILN—inguinal lymph nodes.</p

During gavage infection with <i>S</i>. Typhimurium, bacteria colonize CLN, spleen and thymus at early time points.

<p>C57BL/6 mice were infected with 10⁵<i>S</i>. Typhimurium by gavage. After 2 or 3 days post-infection, mice were sacrificed and organs analyzed for their bacterial loads by plating homogenates on nutrient agar. Data represent mean CFU/organ and SEM of the pooled results from three (day 2) or two (day 3) independent experiments with 4 mice per group. ILN—inguinal lymph nodes; RLN—retropharyngeal lymph nodes; ALN—axillary lymph nodes.</p

Incidence of cervical lymphadenopathy with cervical lymph nodes being the only ones affected (CL) and general lymphadenopathy with lymph nodes other than CLN affected (GL) in children infected with <i>Brucella</i> by ingestion or by contact with animals.

<p>Analysis for significance of differences using Chi-square test yielded Χ² = 5.504 and p = 0.019.</p><p>Incidence of cervical lymphadenopathy with cervical lymph nodes being the only ones affected (CL) and general lymphadenopathy with lymph nodes other than CLN affected (GL) in children infected with <i>Brucella</i> by ingestion or by contact with animals.</p

Incidence of lymphadenopathy in 307 children (up to 14 years) diagnosed for brucellosis and with known routes of infection.

<p>Analysis for significance of differences using Chi-square test yielded Χ² = 5.752 and p = 0.016.</p><p>Incidence of lymphadenopathy in 307 children (up to 14 years) diagnosed for brucellosis and with known routes of infection.</p

Oral infection with <i>B</i>. <i>melitensis</i> induces pro-inflammatory gene expression in the CLN.

<p>C57BL/6 mice were infected with 10⁹<i>B</i>. <i>melitensis</i> per mouse or mock infected by the oral route. At 5 h, 2, 8, 15 or 29 days post-infection, mice were sacrificed, total RNA of the CLN was extracted and analyzed for expression of genes involved in inflammatory responses by reverse transcription real-time PCR. Results are given as fold expression compared to the signal obtained for mock-infected mice. Data represent means and standard deviations of two independent experiments with 3 or 4 mice per group. * p ≤ 0.05 as compared to mock infected expression levels.</p

Oral infection with <i>B</i>. <i>melitensis</i> results in CLN granuloma formation.

<p>Thin sections of cervical lymph nodes from mice orally infected with 10⁹<i>B</i>. <i>melitensis</i> per mouse for (A) 2, (B) 8, (C) 15, or (D) 29 days were stained with eosin-hematoxylin. (E) and (F) show higher magnifications from day 15 and 29, respectively. White arrowheads mark granulomatous structures that (E) develop as multifocal loose cell arrangements that (F) gradually solidify into compact granulomas composed of epithelioid cells with occasional multinucleated giant cells and few neutrophils.</p