Tracking a Live Yersinia pestis Infection with Bioluminescence

The day has finally arrived when an experimental infection can be tracked real-time over the entire course of the infection. Developing a natural history of a rapidly lethal infectious disease has been a challenge because individual variation clouds the progression and individuals can only be studied after death.

The traditional method to study these infections involves infecting many animals so that cohorts of animals can be sacrificed at set time points, have their organs harvested and bacterial load of the organ determined. Some of the flaws of this method are that the right organs may not be selected to survey and individual variation in infection progression means that wide variation may be found at the time points.

Tracking the progression of an infection with bioluminescence allows the infection to run its full course within each experimental animal, rather than taking cohorts at time points. Elizabeth Carniel’s lab at the Institut Pasteur in Paris created bioluminescent Yersinia pestis, and after doing all the controls to ensure a consistent signal under all growth conditions, demonstrated that a live bubonic plague case can be tracked real-time until  death .

In the first figure below the mouse was sacrificed before septicemia set in to correlate the external signals with specific organs. The bioluminescent Y. pestis was injected at the midline near the navel into the linea alba, a tendon-like covering of the abdominal muscles, to simulate a flea bite. Signals represent the injection site, lymph nodes, and the spleen and liver.

Identifying the bioluminescent signals by dissection. 1. axillary lymph nodes, 2. liver, 3. injection site, 4. inguinal lymph node, and 5. spleen. (Nham et al, 2012)

For 74% of the animals injected, the infection followed the same spread pattern. In all the animals the injection site was lit up from the first day. From there it spread to the inguinal lymph node and then surprisingly to the axillary lymph node. The signal then concentrated in the spleen and liver before it becomes completely systemic. Nham et al (2012) note that the signal completely covers the animal from the their ears to the tails. Confirmation of septicemia came from Y. pestis isolated from blood after the death of the animal. Death occurred on average by the sixth day, coming very quickly after septicemia.

Progression of a bubonic plague infection (Nham et al, 2012)

Some of the mice provided clues on how the bioluminescence jumped from the inguinal node to a axillary node. As the linear glow below suggests, they found a lymph vessel that connects the inguinal node to the axillary node. It is consistent with Y. pestis spreading to linked lymph nodes. Confirmation of the lymph vessel linking the inguinal and axillary nodes is shown by bioluminescence and by dye injected dissected mouse in the figure below.

Identification of the lymph vessel connecting inguinal and axillary lymph nodes. (Nham et al, 2012)

The signal next appears under the diaphragm in areas consistent with the liver and spleen (shown in the first figure). This would not necessarily be a direct line from the axillary lymph node to the spleen and liver but that the spleen and liver became infected at about the same time as the axillary node. Colonization of the liver and spleen are related to their blood clearing functions and are indications of a very early phase of septicemia too low to yield systemic bioluminescence.  Death occurred on average by the sixth day, coming very quickly after septicemia.  Nham et al (2012: 5) report that their “results revealed two important phenomena: (i) the variations in the kinetics of bacterial spread were essentially attributable to the length of time the signal remained limited to the injection site, and (ii) as soon as the signal reached lymph nodes, the disease progressed very rapidly, leading to the animal death within two days.” Time between the injection site and first lymph node varied from one to seven days.

The 26% of mice that did not follow this pattern died rapidly with symptoms of a direct septicemic infection (skipping lymph node signals entirely). This suggests that they either hit a blood vessel at the injection site or may have penetrated the abdominal cavity with the injection.

This study is an important first step in developing the method. From here there are many studies that could be done including the effect of changing individual genes on virulence and progression.

Reference:

Nham, T., Filali, S., Danne, C., Derbise, A., & Carniel, E. (2012). Imaging of Bubonic Plague Dynamics by In Vivo Tracking of Bioluminescent Yersinia pestis PLoS ONE, 7 (4) DOI: 10.1371/journal.pone.0034714

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