Michael G. Pappas

Susceptibility of inbred mice to Leishmania tropica infection: Correlation of susceptibility with in vitro defective macrophage microbicidal activities☆

Eleven mouse strains were inoculated in footpads with amastigotes of Leishmania tropica and observed for 12 weeks. Liver and spleen impression smears from infected mice were examined for the presence of intracellular parasites. Four strains (BALB/cJ, C57L/J, NZW/N, and P/J) failed to heal the subcutaneous lesion and showed evidence of systemic infection; the remaining seven strains (A/J, C3H/HeJ, C3H/HeN, C3HeB/FeJ, C57BL/6J, C57BL/10J, and C57BL/10ScN) were each resistant to infection and resolved their lesions by Week 10. Macrophages from the four susceptible strains could not be activated to kill L. tropica amastigotes by treatment with soluble lymphocyte products in vitro. In contrast, macrophages from all seven resistant strains responded to lymphokine treatment and eliminated 80-90% of intracellular parasites. These results suggest that in vitro macrophage microbicidal activities predict the course of systemic leishmanial disease.

Recent applications of the Dot-ELISA in immunoparasitology.

The dot enzyme-linked immunosorbent assay (Dot-ELISA) is a highly versatile solid-phase immunoassay for antibody or antigen detection. The assay uses minute amounts of reagent dotted onto solid surfaces such as nitrocellulose and other paper membranes which avidly bind proteins. After incubation with antigen-specific antibody and enzyme-conjugated anti-antibody, the addition of a precipitable, chromogenic substrate causes the formation of a colored dot on the solid phase which is visually read. The Dot-ELISA has been used extensively in the detection of human and veterinary protozoan and metazoan parasitic diseases, including amebiasis, babesiosis, fascioliasis, cutaneous and visceral leishmaniasis, cysticercosis, echinococcosis, malaria, schistosomiasis, toxocariasis, toxoplasmosis, trichinosis, trypanosomiasis and even ixodid tick infestation. The technique is rapid, easy to perform and interpret, reagent conservative, cost effective and field portable. In addition, the Dot-ELISA may be configured to detect antibodies or parasite antigen in either microtiter plates for large-batch testing or with dipsticks for small numbers of determinations. A slight modification of the Dot-ELISA procedure allows the determination of infection rates of vectors such as ticks and sandflies with parasites.

Leishmania mexicana: Purine metabolism in promastigotes, axenic amastigotes, and amastigotes derived from vero cells

Leishmania mexicana mexicana promastigotes, axenic amastigotes, and amastigotes derived from Vero cells were examined for de novo purine synthesis and mechanisms of purine salvage. Both promastigotes and axenic amastigotes were incapable of de novo purine synthesis, as shown by the lack of [14C]formate and [14C]glycine incorporation into purine nucleotide pools. However, the ready incorporation of [14C]hypoxanthine, [14C]adenine, and [14C]guanine suggested that purine salvage pathways were operating. In addition, a significant percentage (greater than or equal to 60%) of the total label from these purine precursors was associated with adenylate nucleotides. Nucleotide pool levels of axenic amastigotes were consistently greater but the specific activities were less than those of promastigotes, suggesting a slower rate of purine metabolism in the axenic amastigote form. Similar results were obtained from amastigotes isolated from infected Vero cells.

Reduced false positive reactions in the Dot-enzyme-linked immunosorbent assay for human visceral leishmaniasis.

Specificity of the Dot-enzyme-linked immunosorbent assay (Dot-ELISA) for visceral leishmaniasis was significantly improved through the use of enzyme-conjugated antisera specific for IgG heavy chains. Of sera from Kenyans with visceral leishmaniasis, 97% (29/30) were positive using horseradish peroxidase (HRP)-conjugated anti-IgG (heavy and light chain specific) which detected bound IgG and IgM. False positive reactions occurred in 80% of sera from both trypanosomiasis-infected patients (8/10) and apparently healthy Africans (24/30). HRP-conjugated anti-IgG (heavy chain specific), which detected only bound IgG, significantly reduced false positive reactions among trypanosomiasis-infected (2/10, P less than 0.02) and healthy Africans (6/30, P less than 0.001), without reducing test sensitivity in leishmaniasis patients. No false positives occurred when either HRP-conjugated antiserum was used to assay sera from 30 North Americans. Application of enzyme-conjugated antisera specific for IgG improves the serodiagnostic value of the Dot-ELISA for individual patient evaluation and epidemiologic investigations.

Determination of IgM and IgG antibodies to Toxoplasma using the IFA test, ELISA, and Dot-ELISA procedures

The dot enzyme-linked immunosorbent assay (Dot-ELISA) and the enzyme-linked immunosorbent assay (ELISA) were compared with the immunofluorescent antibody test (IFA) for detection of IgM- and IgG-specific antibodies to human toxoplasmosis. Reciprocal titers were determined in all three assays using sera from 56 patients with suspected toxoplasmosis or with symptoms and diseases requiring exclusion of toxoplasmosis and control sera from 56 healthy persons. Using the Dot-ELISA, six patient sera (10.7%) were positive at titers of greater than equal to 1024 for IgM antibodies (titer range 1024-16 384) and 47 sera (84%) were positive for IgG antibodies (titer range 16-262 144) at a titer of greater than or equal to 16. One control serum was reactive for IgM (titer 1024) and 10 control sera (18%) were positive for IgG in the Dot-ELISA. In the ELISA, at titers of greater than or equal to 128, five sera (9%) were reactive for IgM (titer range 128-512) and 52 sera (92.8%) were reactive for IgG (titer range 32-8192) at a titer of greater than or equal to 32; no control sera gave positive reactions for IgM while 10 sera (18%) were positive for IgG in the ELISA. Using the IFA test at reciprocal titers of greater than or equal to 16, four sera (7.1%) were positive for IgM (titer range 32-512), and 51 sera (91%) were positive for IgG (titer range 16-8192). None was reactive for IgM, and eight sera (14%) were positive for IgG (titer range 32-128) in the IFA test. The Dot-ELISA correlated well with the IFA test (correlation coefficient = 0.895) and the ELISA correlated slightly higher with the IFA test (correlation coefficient = 0.910) for detection of IgG antibodies to Toxoplasma gondii.

Antileishmanial activities of macrophages from C3H/HeN and C3H/HeJ mice treated with Mycobacterium bovis strain BCG.

C3H/HeN and C3H/HeJ mice were infected ip with viable BCG, a macrophage-activating agent, and their peritoneal exudate macrophages exposed to Leishmania tropica amastigotes. Macrophages from BCG-infected C3H/HeN mice had both leishmanicidal activities described for lymphokine activation of C3H/HeN macrophages in vitro: increased resistance to L. tropica infection, followed by intracellular killing of the parasite. Macrophages from BCG-infected C3H/HeN mice were also activated to kill tumor cells in vitro. In contrast, macrophages from BCG-treated C3H/HeJ mice were not resistant to L. tropica infection, did not kill intracellular amastigotes over 72 hr in culture, and were not cytotoxic to tumor cells.

DESTRUCTION OF LEISHMANIA

Publisher Summary The Leishmania are protozoan parasites that exist in two discrete forms: The motile promastigote is found in the insect vector and this form converts to a nonmotile amastigote form in vertebrate hosts. A recent examination of culture conditions necessary for the multiplication of Leishmania tropica in vitro suggests that the adherence of macrophages may alter the physiology of the cell, inducing an environment hostile for survival and/or multiplication of the parasite. Macrophage cultures treated with lymphokines before infection induce an immediate loss of infectivity of Leishmania for the activated macrophage population. A number of similarities exist in the activation of macrophages for killing of Leishmania and for killing of rickettsiae. As with all assays of macrophage activation, there are advantages and disadvantages of using Leishmania as the target for macrophage-mediated killing. The propagation of amastigotes of L. tropica and other species causing cutaneous leishmaniasis in footpads of BALB/c mice is a convenient and reliable way of maintaining a stock of amastigotes for in vitro use.

Intracellular Destruction of Leishmania Tropica by Macrophages Activated in Vivo with Mycobacterium Bovis Strain BCG

Leishmania tropica, an intracellular protozoan parasite, infects and replicates in macrophages of C3H/HeN and C3H/HeJ mice in vitro (1). Exposure of resident peritoneal macrophages to amastigotes for 1 hr results in 20–25% infected macrophages, Intracellular parasites increase from 1.7 to >3.5 per macrophage over 72 hr.

Evaluation of complement fixation procedures for the diagnosis of visceral leishmaniasis

Three complement fixation (CF) procedures were evaluated for their ability to detect serum antibodies to visceral leishmaniasis. These tests differ in their use of buffers, volumes of complement and sensitized erythrocyte concentrations, incubation times and percentage haemolysis endpoints. Freeze-thawed sonicates of Leishmania donovani promastignotes were used as antigen. Test sensitivity was determined using sera from 46 Kenyans with parasitologically proven leishmaniasis. The frequencies of positive reactions in all three tests were 96-97% and positive antibody titres ranged from 1:16 to 1:4096. Specificity was determined with 20 sera from healthy individuals with no known exposure to leishmaniasis. The frequencies of false positive reactions were 0-10% in the control sera, with titres up to 1:16. No cross-reactions were observed with sera from patients with bacterial, fungal and other parasitic diseases. In replicate experiments, 99-100% of the sera tested were within one titre dilution of each other. All three CF procedures provide very good sensitivity, specificity and low cross-reactivity and are statistically similar in their capacity to diagnose visceral leishmaniasis.

In vitro macrophage antimicrobial activities and in vivo susceptibility to Leishmania tropica infection.

L. tropica, an obligate intracellular parasite, replicates in phagolysosomes of macrophages. Resistance to L. tropica infection may depend upon alteration of this intracellular environment (1,2). Soluble products of antigen- or mitogen-stimulated lymphocytes (lymphokines) induce enhanced macrophage antimicrobial activity against a number of intracellular organisms (3,4,5,6,7). These activated macrophages may be effector cells during resolution of L. tropica infections. In this study we analyzed the interaction of L. tropica and macrophages treated with lymphokines in vitro, and correlated these findings with susceptibility to L. tropica infection in vivo.