The SCID Mouse Model for Identifying Virulence Determinants in Coxiella burnetii

Abstract

Coxiella burnetii is an intracellular, zoonotic pathogen that is the causative agent of Q fever. Infection most frequently occurs after inhalation of contaminated aerosols, which can lead to acute, self-limiting febrile illness or more serve chronic infections such as hepatitis or endocarditis. Macrophages are the principal target cells during infection where C. burnetii resides and replicates within a unique phagolysosome-like compartment, the Coxiella-containing vacuole (CCV). The first virulence determinant described as necessary for infection was full-length lipopolysaccarride (LPS); spontaneous rough mutants (phase II) arise after passage in immuno-incompetent hosts. Phase II C. burnetii are attenuated in immuno-competent animals, but are fully capable of infecting a variety of host cells in vitro. A clonal strain of the Nine Mile isolate (RSA439, clone 4), has a 26 KDa chromosomal deletion that includes LPS biosynthetic genes and is uniquely approved for use in BL2/ABL2 conditions. With the advances of axenic media and genetic tools for C. burnetii research, the characterization of novel virulence determinants is ongoing and almost exclusively performed using this attenuated clone. A major problem with predicting essential virulence loci with RSA439 is that, although some cell-autonomous phenotypes can be assessed in tissue culture, no animal model for assessing pathogenesis has been defined. Here we describe the use of SCID mice for predicting virulence factors of C. burnetii, in either independent or competitive infections. We propose that this model allows for the identification of mutations that are competent for intracellular replication in vitro, but attenuated for growth in vivo and predict essential innate immune responses modulated by the pathogen during infection as a central pathogenic strategy.

Keywords: C. burnetii; SCID mouse; animal model; transposon mutagenesis; virulence.

Figure 1

Time course of intra-peritoneal challenge of C57/BL6 mice with NMII and the specific IFN-γ CD4+ T-cells response. (A) Splenomegaly calculated as spleen weight as a percentage of total body weight at the time of necropsy on days 3, 7, 14, and 28 after infection with 1 x 10⁶ GE of NMII, or at 3 days after infection with 1 x 10⁶ with dotA NMII mutant. (B) IFN-γ ELIspot was performed using CD4+ T cells purified from NMII infected spleens at 28 days after infection and stimulated with naïve splenocytes loaded with formalin-fixed NMI (WCVI), or stimulated with concavalin A (ConA). Data are represented as spot forming cells per 1 × 10⁶ cells after deduction of the background spots counted in un-stimulated control wells. (C) Genome equivalents calculated using TaqMan real-time PCR with DNA purified from infected lungs from 5 mice on days 3, 7, 14, and 28 days after infection with 1 × 10⁶ GE of NMII or at 3 days after infection for 1 × 10⁶ with dotA NMII mutant. (D) Genome equivalents calculated using TaqMan real-time PCR with DNA purified from infected spleens from 5 mice on days 3, 7, 14, and 28 days after infection with 1 × 10⁶ GE of NMII or at 3 days after infection for 1 × 10⁶ with dotA NMII mutant. For all panels, error bars represent standard deviations from the mean.

Figure 2

Infection of SCID mice with NMII is dose-responsive after intra-peritoneal challenge. (A) Genome equivalents per organ were calculated using TaqMan real-time PCR with DNA purified from infected spleens of 3 mice on days 10 or 14 after intra-peritoneal challenge with 1 × 10⁵ to 1 × 10⁸ GE of NMII. The R² (goodness-of-fit) for the linear regression was 0.86 calculated using Prism GraphPad software. (B) Genome equivalents per organ were calculated using TaqMan real-time PCR with DNA purified from infected lungs of 3 mice on days 10 or 14 after intra-peritoneal challenge with 1 × 10⁵ to 1 × 10⁸ GE of NMII. (C) Genome equivalents per organ were calculated using TaqMan real-time PCR with DNA purified from infected hearts of 3 mice on days 10 or 14 after intra-peritoneal challenge with 1 × 10⁵ to 1 × 10⁸ GE of NMII (D) Splenomegaly calculated as spleen weight as a percentage of total body weight at the time of necropsy on days 10 or 14 after infection with 1 × 10⁵ to 1 × 10⁸ GE of NMII.

Figure 3

Gross Pathology of SCID mouse organs after intra-peritoneal challenge. SCID mice were challenged with 1 × 10⁸ GE or PBS via intra-peritoneal route and sacrificed 10 days after challenge. Spleens and livers were removed from challenged and control mice. Arrows indicate spots of gross pathology on the livers of mice after challenge with NMII.

Figure 4

Intra-peritoneal challenge of SCID mice can be used to test the virulence of NMII mutants. (A) Genome equivalents calculated using TaqMan real-time PCR with DNA purified from infected spleens of 5 mice per group on day 14 after challenge with 1 × 10⁶ GE equivalents of the strains shown. (B) Genome equivalents calculated using TaqMan real-time PCR with DNA purified from infected lungs of 5 mice per group on day 14 after challenge with 1 × 10⁶ GE equivalents of the strains listed in the figure legend. (C) Splenomegaly calculated as spleen weight as a percentage of total body weight at the time of necropsy on day 14 after infection with 1 × 10⁶ GE equivalents of the strains listed in the figure legend. For all panels, the data was analyzed using One-way ANOVA followed by the Dunnett's multiple comparisons test against NMII with Prism GraphPad software and significance is displayed using ^* for P < 0.05, ^*** for P < 0.005, and ^**** for P < 0.0001. Error bars represent standard deviations from the mean.

Figure 5

Fitness of dotA and CB0206 Himar1 transposon mutants relative to NMII Coxiella burnetii. The relative fitness of dotA and CB0206 mutants was compared to wild-type NMII in mixed infections in 5 mice per group where NMII and mutant were inoculated into SCID mice IP in equal proportions. The competitive index (CI) for each infection was calculated by dividing the ratio of mutant-to-wild type genomes in the output sample by that in the inoculum. A CI of 1 indicates the mutant strain has no loss of fitness relative to wild-type bacteria. The data was analyzed using One-way ANOVA followed by the Dunnett's multiple comparisons test against NMII with Prism GraphPad software and significance is displayed using ^* for P < 0.05. Error bars represent standard deviations from the mean.

Figure 6

Large particle aerosolization (LPA) intra-tracheal challenge of SCID mice with NMII. (A) Colony forming units of NMII present in the inoculum before and after aerosolizaiton with a MicroSprayer® Aerosolizer (Penn-Century Inc.) as determined by spot plating on ACCM-2. (B) Splenomegaly expressed as spleen weight as a percentage of total body weight at the time of necropsy on days 5, 7, 10 after intra-tracheal challenge with 1 × 10⁶ GE of NMII. (C) Genome equivalents per organ were calculated using TaqMan real-time PCR with DNA purified from infected spleens of 5 mice per group on days 5, 7, and 10 after intra-tracheal challenge with 1 × 10⁶ GE of NMII. For all panels, error bars represent standard deviations from the mean.