Exploration of refined humane endpoints for melioidosis in BALB/c mice

lab animal Article https://doi.org/10.1038/s41684-025-01667-5 Exploration of refined humane endpoints for melioidosis in BALB/c mice Check for updates Michael P. Harris1, Kay B. Barnes1, Thomas R. Laws1, Emily May1, Michelle Nelson1, Sarah V. Harding1,2 & Thomas C. Maishman    1 The development of humane endpoints is critical for refining scientific studies involving animals. Body weight and clinical signs of disease data collected in four recent studies assessing medical countermeasures for utility against the disease melioidosis in mice were further analyzed. Here we used this information to ascertain whether a suitable alternative humane endpoint could be identified. A total of 66 possible alternative humane endpoints were explored, which varied the threshold values of the ‘percentage body weight loss post-challenge’ and ‘the clinical signs over time’ following cessation of treatment. The findings indicated a suitable alternative endpoint of a percentage weight loss threshold of 25%, and/or using an average total clinical signs score ≥5 over a 48-h period. This endpoint resulted in a sizeable reduction in median ‘sign-days’ (total clinical score multiplied by the number of days remaining in study) per mouse of 21 days (ranging from 8 to 56 between studies), while maintaining 100% sensitivity and 93% specificity (ranging from 79% to 97% between studies). In addition, the risk of altering the scientific outcome of each study remained low when utilizing this new endpoint. In conclusion, current humane endpoints in this setting can be refined without negatively impacting the key study findings. Melioidosis is a disease caused by the bacterium Burkholderia pseudomallei, a Gram-negative organism found in tropical and subtropical regions of the world1. The global burden of human melioidosis is substantial, with an estimated 165,000 cases worldwide and 89,000 deaths per year2. B. pseudomallei is intrinsically resistant to many antimicrobials and has a large range of virulence factors allowing it to avoid the host immune response, therefore making it challenging to treat3. Currently, the recommended treatment consists of intravenous antibiotics for 10–14 days, followed by an oral eradication phase, with a total treatment duration of 20 weeks4. Despite successful completion of the antibiotic regimen, relapse of infection can occur in up to 23% of cases and is associated with a mortality similar to that of the initial infection5. It is therefore essential that alternative treatments or treatment strategies are investigated. For the use of new treatments in humans, regulatory authorities currently require that they be demonstrated to be safe and effective. Preclinical evaluation of these new treatments involves the use of animal models to demonstrate efficacy. To be able to evaluate new treatments effectively, animal infections that model human disease are necessary. Rodent models of melioidosis are well described6–9, and mouse models have been extensively used to characterize the pathogenesis of melioidosis. The presentation of disease depends not only on the route of infection but also on the strain of the mouse. BALB/c mice are more susceptible to infection with B. pseudomallei and represent an acute model of melioidosis, whereas C57BL/6 mice are more resistant and may represent a more chronic model of disease6. Despite the infection in the BALB/c mouse model being acute, it is considered an appropriate model for evaluating the efficacy of antibiotics8. Studies have demonstrated that BALB/c mice infected with B. pseudomallei can be effectively treated with antibiotics, with 100% survival at the end of the treatment period and no detection of bacteria within their organs10,11. However, relapse to infection is often observed following the cessation of antibiotic therapy10,11. Relapse is usually observed from 7 days after the cessation of therapy, with weight loss most commonly observed first, followed by the development of clinical signs of disease, which gradually increase until a humane endpoint is reached. In four recent studies, named study 111, study 2 (unpublished), study 312 and study 413, antibiotics were evaluated as monotherapies (finafloxacin, doxycycline or co-trimoxazole) and as combinations (finafloxacin in combination with doxycycline and finafloxacin in combination with a capsular conjugate vaccine). All four studies used the same primary outcome measure: time to lethal endpoint. In addition to protection being the primary parameter measured, additional data were collected on body weight and clinical signs of disease. Although these mice were euthanized by cervical dislocation once they reached their predefined humane endpoint (as required under the Animals Scientific Procedures Act14), 1 Defence Science and Technology Laboratory, Porton Down, Salisbury, UK. 2Respiratory Sciences, University of Leicester, Leicester, UK. e-mail: Lab Animal | Volume 55 | February 2026 | 40–47 40 Article https://doi.org/10.1038/s41684-025-01667-5 Table 1 | Summary of data collected from each of the four studies Parameter Study 111 Study 2a Study 312 Study 413 Number of mice 105 110 135 206 Number (%) reaching predefined end of study 67 (63.8%) 92 (83.6%) 116 (85.9%) 123 (59.7%) Number (%) reaching humane endpoint 34 (32.4%) 16 (14.5%) 19 (14.1%) 76 (36.9%) Number (%) succumbing to infection before being euthanized 4 (3.8%) 2 (1.8%) 0 (0%) 7 (3.4%) Study duration (post-challenge in days) 66 53 43 36 Number of treatment comparison groups 3 2 4 3 106 Challenge dose (mean retained dose in CFU) 142 62 100 Treatment duration (days) 14 14, 14b 14 7 Treatment start time (h) 24 24 24 or 36 36 or 48 a Manuscript in preparation; b14 days followed by 14 days ‘pause’, followed by a further 14 days. some animals still succumbed to infection (Table 1). Alternative humane endpoints could therefore be explored to both reduce the likelihood of animals succumbing to infection before euthanasia and minimize the potential suffering of those exhibiting clinical signs. This refinement is an important component of the 3Rs (replacement, reduction and refinement) principles to “minimize the pain, suffering, distress or lasting harm that may be experienced by research animals, and which improve their welfare”15. A preliminary analysis, in the form of a week-long hackathon involving individuals from a variety of disciplines, took place in January 2023. The aim of the hackathon was to try to identify which parameters were most closely associated with the animals (mice) that succumbed to infection by B. pseudomallei. A wide range of approaches was explored to address this question, including network analyses, decision trees, random forests, recurrent neural networks and a review of the existing literature. The findings indicated that two of the most prominent parameters were consecutive percentage body weight loss post-challenge and the total c (...truncated)