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.

In BALB/c Mice, IL-4 Production During the Initial Phase of Infection withLeishmania majorIs Necessary and Sufficient to Instruct Th2 Cell Development Resulting in Progressive Disease

“…1). Furthermore, the numbers of viable parasites recovered after culture in vitro of footpad tissue under limiting dilution condition (32) confirmed that although parasite growth was controlled in V␤4 CD4 ϩ T cell-deficient BALB/c mice comparably to that in resistant C57BL/6 mice (ϳ200 parasites/footpad) 6 wk after infection, footpad lesions of V␤4 CD4 ϩ T cell-deficient BALB/c mice treated with IL-4 during the first 64 h of infection contained numbers of parasites (ϳ10 8 ) similar to those found in control BALB/c mice.…”

Neutrophils and Macrophages Cooperate in Host Resistance againstLeishmania braziliensisInfection

“…Lesion size was monitored on a weekly basis for 10 wk using a digital caliper (Thomas Scientific). Parasite load was determined using a quantitative limiting dilution assay as described previously (18).…”

Immune response to Leishmania (Leishmania) chagasi infection is reduced in malnourished BALB/c mice

“…A fragment from each organ was collected to be used for parasite quantification. Quantitative limiting dilution culture was performed as described by Titus et al (1985) and modified by Marques-da-Silva et al (2005). One weighed fragment from each organ (liver and spleen) was homogenized in a tissue grinder and suspended in 500 μL of Grace's Insect Medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% heat-inactivated foetal calf serum (FCS) (Cripton, Andradina, SP, Brazil), 2 mM L-glutamine (Gibco BRL) and 100 U/mL penicillin G potassium (USB Corporation, Cleveland, OH, USA), pH 6.5 in 96-well flatbottom microtitre plates (160 μL per well).…”