A limiting dilution assay for quantifying Leishmania major in tissues of infected mice

Abstract

A limiting dilution assay for the quantification of Leishmania major in infected mouse tissue was developed. The assay was found to be both sensitive and reliable, and, due to its design, could be scored either visually or following the incorporation of 3H-thymidine by the growing parasites. Results are presented in which the assay was employed to enumerate L. major in the tissues of susceptible (BALB/c) and resistant (CBA) mice at intervals after infection with L. major. It was found that parasites could be detected at the site of injection with L. major as early as 3 days after infection. By day 8, a substantial increase in the number of parasites at the lesion site had occurred in both strains of mice. Subsequently, whereas the number of parasites decreased in the lesions of CBA mice, their number steadily increased in the lesions of BALB/c mice. Parasites were detected in lymph nodes draining the lesion site in both BALB/c and CBA mice by 28 days after infection. Interestingly, a low number of L. major was found in the lymph nodes of CBA mice at 100 days after infection, a time when no parasites could be detected at the lesion site. Previous results from this laboratory have demonstrated that the adoptive transfer of L. major-specific L3T4-positive T-cell populations exacerbated cutaneous lesions induced by L. major in BALB/c mice. Experiments presented here indicate that the adoptive transfer of L. major-specific T-cells also exacerbated cutaneous leishmaniasis in CBA mice. Using the sensitive limiting dilution assay presently described, it was found that this unexpected exacerbative effect of L. major-specific T-cells on lesion development was accompanied by a substantial increase in the number of parasites in the lesions of the adoptively transferred mice.