Oropharyngeal aspiration of Burkholderia mallei and Burkholderia pseudomallei in BALB/c mice

Abstract

Burkholderia mallei and Burkholderia pseudomallei are potentially lethal pathogens categorized as biothreat agents due, in part, to their ability to be disseminated via aerosol. There are no protective vaccines against these pathogens and treatment options are limited and cumbersome. Since disease severity is greatest when these agents are inhaled, efforts to develop pre- or post-exposure prophylaxis focus largely on inhalation models of infection. Here, we demonstrate a non-invasive and technically simple method for affecting the inhalational challenge of BALB/c mice with B. pseudomallei and B. mallei. In this model, two investigators utilized common laboratory tools such as forceps and a micropipette to conduct and characterize an effective and reproducible inhalational challenge of BALB/c mice with B. mallei and B. pseudomallei. Challenge by oropharyngeal aspiration resulted in acute disease. Additionally, 50% endpoints for B. pseudomallei K96243 and B. mallei ATCC 23344 were nearly identical to published aerosol challenge methods. Furthermore, the pathogens disseminated to all major organs typically targeted by these agents where they proliferated. The pro-inflammatory cytokine production in the proximal and peripheral fluids demonstrated a rapid and robust immune response comparable to previously described murine and human studies. These observations demonstrate that OA is a viable alternative to aerosol exposure.

Figure 1. Challenge by oropharyngeal aspiration.

Mice were lightly anesthetized as described in the materials and methods and manually restrained in an upright position (A). Small curved forceps were applied to gently open the mouth and secure the tongue to the lower jaw (A, inset). (B) 30 µl of inoculum was administered to the back of the mouth using a pipette and sterile tip. The nares were blocked by the second investigator (C) to prevent obligate nasal breathing and compel inhalation of the inoculums.

Figure 2. The 50% lethal dose of B. pseudomallei K96243 when administered by oropharyngeal aspiration.

Five doses of 10-fold serial dilutions were prepared to administer 4.06×10⁴ cfu, 3.83×10³ cfu, 640 cfu, 40 cfu and 4 cfu of B. pseudomallei K96243 to BALB/c mice by OA as described as described in the materials and methods and Fig. 1. Mice were observed for 14 days. The actual inhaled doses are shown in the graph and were utilized to calculate LD₅₀ (3.11 cfu).

Figure 3. Reproducibility of oropharyngeal aspiration.

The results of four independent challenges are presented here. In each case, 15 BALB/c mice were challenged by OA with B. pseudomallei K96243 and the inhaled dose was determed as described in the materials and methods section. Asterics identify challenges performed for this work (* from model, ** from LD₅₀).

Figure 4. The 50% lethal dose of B. mallei ATCC 23344 when administered by oropharyngeal aspiration.

Five doses of 10-fold serial dilutions were prepared to administer 3.8×10⁵ cfu, 3.6×10⁴ cfu, 3.36×10³ cfu, 366 cfu and 36 cfu of B. mallei ATCC 23344 to BALB/c mice by OA as described as described in the materials and methods and Fig. 1. Mice were observed for 14 days. The actual inhaled doses are shown in the graph and were utilized to calculate LD₅₀ (1.7×10³ cfu).

Figure 5. Systemic murine melioidosis resulting from oropharyngeal aspiration of B. pseudomallei.

BALB/c mice were challenged with approximately 130 cfu (inhaled dose) of B. pseudomallei K96243. A group of ten mice were observed for survival analysis (A) over the duration of the experiment. At 24 hour increments, three mice were randomly selected and placed under deep Ketamine/Xylazine anesthesia. They were weighed (B), and their blood (C), spleens (D), livers (E) and lungs (F) were harvested for bacterial burden assessment. Each graph (B-F) is presented as the average cfu per gram of tissue (or ml of blood) of the three mice ± SEM. CFUs at zero hour in F (*) indicate challenge dose.

Figure 6. Lymphocyte infiltration of lungs following oropharyngeal aspiration of B. pseudomallei.

At 24 hour increments, five mice were randomly selected and bronchoalveolar lavage fluid was collected. The number of lymphocytes/BALF were assessed as a measurement of pulmonary inflammation and are presented as the average of the five counts with the SEM presented on the graph. Controls depict lymphocytes from uninfected naïve mice while sham challenged (PBS) samples were harvested 72 hours following inhalation to reflect the timepoint where signs of inflammation were maximal in our studies.

Figure 7. Proinflammatory cytokine production in the proximal lung fluids following oropharyngeal aspiration challenge with B. pseudomallei.

Bronchoalveolar lavage fluid collected from OA-challenged mice (n = 5) were analyzed by cytometric bead analysis for the presence of proinflammatory cytokines. Each assay was performed in triplicate. The average of the five mice is shown in pg/ml with the SEM displayed on the graph. *indicates statistical significance compared to naïve BALF as determined by one-way Anova with Tukey's post-test (*<0.05, **<0.01).

Figure 8. Proinflammatory cytokine concentration in the peripheral blood following oropharyngeal aspiration challenge with B. pseudomallei.

Serum from each of the mice (n = 5) examined above was analyzed by cytometric bead analysis for the presence of proinflammatory cytokines. Each assay was performed in triplicate. The average of the five mice is shown in pg/ml with the SEM displayed on the graph. *indicates statistical significance compared to naïve serum as determined by one-way Anova with Tukey's post-test (*<0.05, **<0.01, ***<0.001).

Figure 9. Inflammatory lung scores following OA-challenge.

B. pseudomallei or PBS was delivered to BALB/c mice by OA. The lungs were collected and processed as described above and alalyzed for signs of perivascular, peribronchial and interstitial inflammation according to previously described methods –. Controls depict lungs from uninfected naïve mice while PBS represents sham challenged tissues. The average score of the five mice is shown with the SEM displayed on the graph. *indicates statistical significance compared to the PBS controls as determined by one-way Anova with Tukey's post-test (*<0.05, ***<0.0001).

Figure 10. Temporal lung lesions induced by B. pseudomallei when administered by oropharyngeal aspiration.

Lungs were collected from mice at 24 h, 48 h and 72 h post infection, stained (H&E stains and Periodic Acid Schiff) and analyzed for B. pseudomallei induced lung damage. Focal pyogranulomatous lesions associated with bronchioles are indicated by arrows. PBS (72 h) depict sham challenged lung tissue at 72 hours following administration of PBS. The data presented here represent a consensus of five lungs examined at each timepoint.