Coxiella burnetii Intratracheal Aerosol Infection Model in Mice, Guinea Pigs, and Nonhuman Primates

Abstract

Coxiella burnetii, the etiological agent of Q fever, is a Gram-negative bacterium transmitted to humans by inhalation of contaminated aerosols. Acute Q fever is often self-limiting, presenting as a febrile illness that can result in atypical pneumonia. In some cases, Q fever becomes chronic, leading to endocarditis that can be life threatening. The formalin-inactivated whole-cell vaccine (WCV) confers long-term protection but has significant side effects when administered to presensitized individuals. Designing new vaccines against C. burnetii remains a challenge and requires the use of clinically relevant modes of transmission in appropriate animal models. We have developed a safe and reproducible C. burnetii aerosol challenge in three different animal models to evaluate the effects of pulmonary acquired infection. Using a MicroSprayer aerosolizer, BL/6 mice and Hartley guinea pigs were infected intratracheally with C. burnetii Nine Mile phase I (NMI) and demonstrated susceptibility as determined by measuring bacterial growth in the lungs and subsequent dissemination to the spleen. Histological analysis of lung tissue showed significant pathology associated with disease, which was more severe in guinea pigs. Infection using large-particle aerosol (LPA) delivery was further confirmed in nonhuman primates, which developed fever and pneumonia. We also demonstrate that vaccinating mice and guinea pigs with WCV prior to LPA challenge is capable of eliciting protective immunity that significantly reduces splenomegaly and the bacterial burden in spleen and lung tissues. These data suggest that these models can have appreciable value in using the LPA delivery system to study pulmonary Q fever pathogenesis as well as designing vaccine countermeasures to C. burnetii aerosol transmission.

Keywords: Coxiella burnetii; MicroSprayer; Q fever; aerosol; intratracheal; large-particle aerosol.

FIG 1

BL/6 mouse response to challenge dose. BL/6 mice were challenged with increasing doses of C. burnetii RSA 493 and monitored for clinical signs of disease over a 14-day period. (A) The degree of body weight loss over the course of the experiment was closely correlated with the infectious dose given. (B) Splenomegaly, as a function of total body weight, increased with increasing doses. (C) Genome equivalents recovered from spleens and lungs of infected mice were reflective of the dose received, which was also consistently higher in lungs than in the spleen.

FIG 2

Comparison of effects of different challenge routes on the dose response in mice. BL/6 mice were challenged with 1 × 10⁵ GE of C. burnetii RSA 493 via the i.p., i.t., or LPA challenge route. (A) Mice were monitored for clinical signs of disease over a 14-day period, including any changes to body weight. (B) Weights of lungs, livers, and spleens relative to body weight were measured at necropsy. (C and D) Tissue samples were also analyzed for histopathological changes by staining spleens, livers, and lungs with hematoxylin and eosin and grading for inflammation. Representative images for five mice per group are shown (magnification, ×10; scale bar, 100 μm). Dashed lines, histiocytic inflammation in red pulp; arrows, perivascular cuffing; circles, hepatic granuloma. Error bars represent the standard error of the mean; asterisks indicate significant differences (*, P < 0.05; **, P < 0.01).

FIG 3

Guinea pig response to challenge dose. Hartley guinea pigs were challenged with increasing doses of C. burnetii RSA 493 and monitored for clinical signs of disease over a 14-day period. (A) Internal temperatures show guinea pigs developing fever when challenged with 1 × 10⁴ and 1 × 10⁵ GE (fever is defined as a 0.5°C temperature increase relative to baseline). (B) Guinea pigs challenged with 1 × 10⁵ GE were the only group not to increase in weight by the end of the study. (C) An increase in challenge dose correlated with an increase in splenomegaly (spleen weight as a percentage of total body weight at necropsy). Error bars represent the standard error of the mean; asterisks indicate significant differences (*, P < 0.05; **, P < 0.01).

FIG 4

Bacterial burden and histological changes in C. burnetii-infected guinea pigs. Hartley guinea pigs were euthanized after a 14-day aerosol infection with C. burnetii RSA 493. (A) Spleen and lung samples were analyzed for C. burnetii numbers by qPCR. Error bars represent the standard error of the mean; asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.005). (B) Lung sections from uninfected guinea pigs (Bi) were compared histologically (H&E staining) with those from guinea pigs infected with 1 × 10⁵ GE (Bii). Infected guinea pigs are characterized by the infiltration of the alveolar spaces by large numbers of foamy macrophages admixed with occasional multinucleated giant cells, neutrophils and lymphocytes, fibrin, and edema (histiocytic interstitial pneumonia; arrows denote leukocyte infiltration) (magnification, ×2; scale bar, 500 μm). (C) Tissue sections were scored histologically for severity of lung inflammation. Data are representative of 3 guinea pigs per group, and significance is measured relative to uninfected animals.

FIG 5

LPA infection of NHPs with C. burnetii. Four NHPs were infected with 1 × 10⁶ GE of C. burnetii RSA493 via the LPA route and monitored for signs of disease over 3 weeks. (A) Telemetric temperature readings indicate normal readings (green dots) for the first 3 days following infection, followed by a febrile response (red dots) lasting 4 to 7 days. (B) A thoracic radiograph (left lateral) taken at 7 days postinfection shows air bronchograms, a moderate alveolar pattern in caudal/dorsal lung fields, and mildly enlarged tracheobronchial lymph nodes. (C) Tissue sections were scored histologically for severity of inflammation. Data are representative of 4 infected NHPs and 2 uninfected NHPs.

FIG 6

Protection against C. burnetii aerosol challenge. BL/6 mice and Hartley guinea pigs were vaccinated s.c. in a prime-boost manner with 10 μg and 50 μg WCVI, respectively. At 7 weeks postvaccination, mice and guinea pigs were challenged via the LPA route with 1 × 10⁶ and 1 × 10⁵ GE of C. burnetii RSA493. (A) Both sets of vaccinated animals showed a significant reduction in both spleen and lung burden. (B) Splenomegaly was also markedly reduced in vaccinated groups compared with unvaccinated animals. (C) Histology of lung tissues from WCV-vaccinated guinea pigs (H&E staining) shows no evidence of inflammation (Ci), compared with sham-vaccinated animals that have a central area of necrosis surrounded by large numbers of macrophages, lymphocytes, heterophils, and plasma cells (Cii) (magnification, ×2; scale bar, 500 μm). (D) Tissue sections were scored histologically for severity of lung inflammation. Data are representative of 5 mice per group and 4 guinea pigs per group.