Study design and ethical statement

Immunization and challenge infections were performed in a hamster model of VL to determine the efficacy of centrin gene-deleted L. mexicana (LmexCen^−/−) parasites as a vaccine against experimental L. donovani infection. All animal experiments in this study were reviewed and approved by the Animal Care and Use Committee of the Center for Biologics Evaluation and Research, U.S. Food and Drug Administration (ASP 1999#23) and the National Institute of Allergy and Infectious Diseases (NIAID) (http://grants.nih.gov/grants/olaw/references/phspolicylabanimals.pdf) under animal protocol LMVR4E. The NIAID DIR Animal Care and Use Program complies with the Guide for the Care and Use of Laboratory Animals and with the NIH Office of Animal Care and Use and Animal Research Advisory Committee guidelines. The housing condition of animals were followed standard guidelines by NIH guidelines for the humane care and use of animals. The body weight, fur and physical appearance was monitored and periodically assessed by the facility veterinarian to assess the development of VL. Sample sizes were calculated as the following: 4 hamsters per group can provide over 80% power to detect a 2-fold difference on parasite load (survival) averaged across the tested time points comparing the hamsters with and without the immunization (α = 0.05 and CV = 30%).