Primary pneumonic plague in the African Green monkey as a model for treatment efficacy evaluation

Abstract

Background: Primary pneumonic plague is rare among humans, but treatment efficacy may be tested in appropriate animal models under the FDA 'Animal Rule'.

Methods: Ten African Green monkeys (AGMs) inhaled 44-255 LD(50) doses of aerosolized Yersinia pestis strain CO92. Continuous telemetry, arterial blood gases, chest radiography, blood culture, and clinical pathology monitored disease progression.

Results: Onset of fever, >39°C detected by continuous telemetry, 52-80 hours post-exposure was the first sign of systemic disease and provides a distinct signal for treatment initiation. Secondary endpoints of disease severity include tachypnea measured by telemetry, bacteremia, extent of pneumonia imaged by chest x-ray, and serum lactate dehydrogenase enzyme levels.

Conclusions: Inhaled Y. pestis in the AGM results in a rapidly progressive and uniformly fatal disease with fever and multifocal pneumonia, serving as a rigorous test model for antibiotic efficacy studies.

Figure 1

One-hour averages of heart rate, respiratory rate and body temperature obtained by continuous telemetry in two representative AGMs X774 and X775 infected at time ‘0’. Tables below the illustration for each vital sign describe for all 10 AGM when the measure became significantly different (repeated measures ANOVA) from each animal's baseline. Baseline data with standard deviation are displayed as 24 h repeats on the figure in order to demonstrate how each animal would look at this time PE if not challenged with Y. pestis.

Figure 2

Y. pestis bacteremia following aerosol challenge in AGM. Collected blood was serially diluted and cultured daily each morning onto TSA for upwards of 4 days post-challenge. Data represent the geometric mean CFU/mL of blood based on the dilution factor and colony counts. The limit of detection (LOD) for this assay was defined as 200 CFU/mL of blood (20 CFU per 100 μL plate inoculum). As shown, certain data points fell below this limit, but were still reported due to the presence of countable Y. pestis colonies.

Figure 3

Y. pestis tissue burden in moribund AGM following aerosol challenge. Select tissue samples were collected at necropsy, homogenized, serially diluted, and cultured onto TSA. Data represent the geometric mean CFU/g of tissue for all study animals on the dilution factor, colony counts and sample weights. TBLN, tracheobronchial lymph node; NL, non-lesioned sample; L, lesioned sample.

Figure 4

Radiological Images of the Chest. Serial chest radiographs from two AGMs obtained prior to exposure (day 0), day 3 PE (approximately 72 h PE), and at the time of moribund necropsy approximately 96 h PE are shown. ‘L’ designates left chest with pleural pressure transducer visible on the left side. AGM X775 displays a clear pulmonary parenchyma on day 0, small periohilar infiltrates in the right middle and right caudal lobes on day 3 PE, and large right middle lobe infiltrate and small right caudal infiltrate at moribund necropsy. AGM X784 displays clear pulmonary parenchyma on day 0, small right cranial and left cranial infiltrates on day 3 PE, and large left cranial, right cranial, left middle, right middle, and right caudal lobe infiltrates at moribund necropsy.

Figure 5

Representative pulmonary histopathology of primary pneumonic Y. pestis infection. A) Low power view of fulminant fibrinosuppurative and hemorrhagic pneumonia in a nodular lesion in an AGM (X784) moribund 4 days post-exposure [bar = 100 μm]. B) High power view from same area as A. Abundant extracellular basophilic small rod-like bacteria are present (small arrows). Macrophages within an edema protein-filled alveolus contain numerous intracellular bacteria (arrowhead) [bar = 10 μm]. C) Low power view from a region of the lung without gross lesions from an AGM (X774) moribund 4 days post-exposure. There is prominent hypercellularity of alveolar septae but no substantive exudates. Bluish, granular material within alveoli is composed of abundant extracellular bacteria [bar = 100 μm]. D) High power view from same region as panel C. Bacteria associated with cell surface of alveolar macrophages (arrowheads) as well as extracellular bacteria (small arrows) [bar = 10 μm].