Mouse Model of Coxiella burnetii Aerosolization

Abstract

Coxiella burnetii is mainly transmitted by aerosols and is responsible for multiple-organ lesions. Animal models have shown C. burnetii pathogenicity, but long-term outcomes still need to be clarified. We used a whole-body aerosol inhalation exposure system to mimic the natural route of infection in immunocompetent (BALB/c) and severe combined immunodeficient (SCID) mice. After an initial lung inoculum of 10(4) C. burnetii cells/lung, the outcome, serological response, hematological disorders, and deep organ lesions were described up to 3 months postinfection. C. burnetii-specific PCR, anti-C. burnetii immunohistochemistry, and fluorescent in situ hybridization (FISH) targeting C. burnetii-specific 16S rRNA completed the detection of the bacterium in the tissues. In BALB/c mice, a thrombocytopenia and lymphopenia were first observed, prior to evidence of C. burnetii replication. In all SCID mouse organs, DNA copies increased to higher levels over time than in BALB/c ones. Clinical signs of discomfort appeared in SCID mice, so follow-up had to be shortened to 2 months in this group. At this stage, all animals presented bone, cervical, and heart lesions. The presence of C. burnetii could be attested in situ for all organs sampled using immunohistochemistry and FISH. This mouse model described C. burnetii Nine Mile strain spread using aerosolization in a way that corroborates the pathogenicity of Q fever described in humans and completes previously published data in mouse models. C. burnetii infection occurring after aerosolization in mice thus seems to be a useful tool to compare the pathogenicity of different strains of C. burnetii.

FIG 1

FISH targeting C. burnetii 16S rRNA. (A1) Controls assayed: L929 cells infected with C. burnetii. Blue indicates the DAPI-stained nuclei of L929 cells. Yellow signal indicates colocalization of the C. burnetii-specific 16S rRNA probe (green) and the universal 16S probe EUB (red). C. burnetii appears as a perinuclear, rounded, and vacuolar signal. Magnification, ×100. (A2) Negative control: SCID mice aerosolized with PBS in the liver. Magnification, ×100. (A3) Negative control: SCID mice aerosolized with PBS in the lung. Magnification, ×100. (B to D) Infected tissues of a SCID mouse at 2 months. DAPI-stained nuclei appear in blue. A positive signal appears in yellow, reflecting colocalization of the C. burnetii-specific 16S rRNA probe (green) and the universal EUB probe (red). (B1) Lung. Alveoli are recognized, as defined by the pneumocytes. (B2) Lung. On the right, C. burnetii can be positively identified, presumably in an alveolar macrophage. A positive signal appears as a rounded perinuclear intracytoplasmic and vacuolar signal. (C1 and C2) Liver. Multiple and diffuse rounded signals are evident. (D1 to D3) Heart. A positive signal can be seen in the pericardium (D1 and D2) and myocardium (D1 and D3).

FIG 2

Changes in spleen weight over time in BALB/c and SCID mice. The asterisk indicates a significant intragroup difference (P < 0.05) from day 3.

FIG 3

PCR results in different organs over time in BALB/c and SCID mice. Asterisks indicate significant intragroup differences (P < 0.05) from day 3.

FIG 4

Representative pathological and immunohistological findings for organs from C. burnetii-infected SCID mice at 2 months p.i. C. burnetii infection via aerosol induced an important macrophage infiltration in tissues, as shown by hematoxylin-eosin-saffron staining (figures on the left). Bacteria were visualized by immunostaining with a polyclonal rabbit anti-C. burnetii antibody used at a dilution of 1:2,000, along with hemalun counterstain (figures on the right). Macrophages were packed with coarse granular immunopositive material. (Lung images) Note the macrophage infiltration of the alveolar walls with several intra-alveolar macrophages easily identified by immunostaining. (Liver images) Macrophage infiltration is randomly distributed in liver parenchyma and mixed with necrotic areas. Note the great number of immunopositive macrophages. (Spleen images) Macrophage infiltration is seen mainly in the red pulp. Note the great number of immunopositive macrophages. (Heart images) Note the areas of macrophage infiltration in the pericardium (arrowhead), the myocardium (star), and a cardiac valve (arrow). (Lymph node and bone marrow images) Note the significant infiltration by numerous and highly immunopositive macrophages.