Hypothermic endpoint for an intranasal invasive pulmonary aspergillosis mouse model

Abstract

Immunocompromised mice were infected intranasally with Aspergillus fumigatus as part of a vaccine efficacy study. Although body temperature was measured throughout the study, a formal evaluation of its usefulness as an endpoint criterion was not performed. We retrospectively evaluated survival data and temperature records to determine whether body temperature can be used as an objective predictor of death and included in the humane endpoint criteria for this mouse model. CF1 mice were immunosuppressed with either cortisone acetate or by treatment with antiGR1 (a neutrophil-depleting antibody) and then intranasally challenged with A. fumigatus. Body temperature was measured by using an infrared noncontact thermometer a maximum of 3 times daily until death or euthanasia. A surface body temperature below 29.0 °C was correlated with a poor chance of survival, and using this cutoff point with signs of morbidity (hunched, ruffled fur, respiratory distress) reliably indicates mice for euthanasia without negatively affecting data collection. Using 2 subsequent readings of less than 31.0 °C as an endpoint would have led to premature euthanasia of only one mouse (2.2%). As a single reading, a body temperature of 28.8 °C had a sensitivity of 92.2% and specificity of 90.9%. Hypothermia proved to be a useful addition to the humane endpoint criteria for this mouse model, and veterinary and research groups should discuss their study needs in relation to animal welfare to best determine the most appropriate means of including this parameter.

Figure 1.

The infrared thermometer (MiniTemp MT4, Raytek, Santa Cruz, CA) and demonstration of the method used to record BT.

Figure 2.

Survival over time after intranasal Aspergillus challenge. All mice challenged with A. fumigatus (both vaccinated and unvaccinated) are included (n = 122).

Figure 3.

Percentage mortality at any given temperature reading. Mice were recorded as ‘dead’ when they were either euthanized before the next temperature recording or were found dead between temperature readings or at the next time point.

Figure 4.

BT (mean ± SE) of mice that survived (n = 45) compared with those that eventually died over the course of the experiment. The number of animals recorded as dead changed as mice were euthanized or found dead during the study (days 0 and 1, n = 77; day 2, n = 68; day 3, n = 24; day 4, n = 6; day 5, n = 5; days 6 and 7, n = 1); there are no error bars for time points 6 and 7 because only one animal is represented at those times. *, Significant (P ≤ 0.03) difference between groups at that time point.

Figure 5.

The sensitivity and specificity associated with using each BT as the diagnostic cut-off were used to construct an ROC curve. These curves are commonly used by the medical community to evaluate diagnostic testing and determine the appropriate values to use to categorize patients as either ‘positive’ (that is, with disease) or ‘negative’ (that is, no disease).^,, In our case, when mice were euthanized or died after a given temperature reading, they were designated as positive. Data from the 72-h time point were used, because they represented the largest number of mice in each designation (that is, positive and negative).