Detection of Leishmania donovani and L. tropica in Ethiopian wild rodents

Human visceral (VL, also known as Kala-azar) and cutaneous (CL) leishmaniasis are important infectious diseases affecting countries in East Africa that remain endemic in several regions of Ethiopia. The transmission and epidemiology of the disease is complicated due to the complex life cycle of the parasites and the involvement of various Leishmania spp., sand fly vectors, and reservoir animals besides human hosts. Particularly in East Africa, the role of animals as reservoirs for human VL remains unclear. Isolation of Leishmania donovani parasites from naturally infected rodents has been reported in several endemic countries; however, the status of rodents as reservoirs in Ethiopia remains unclear. Here, we demonstrated natural Leishmania infections in rodents. Animals were trapped in 41 localities of endemic and non-endemic areas in eight geographical regions of Ethiopia and DNA was isolated from spleens of 586 rodents belonging to 21 genera and 38 species. Leishmania infection was evaluated by real-time PCR of kinetoplast (k)DNA and confirmed by sequencing of the PCR products. Subsequently, parasite species identification was confirmed by PCR and DNA sequencing of the 18S ribosomal RNA internal transcribed spacer one (ITS1) gene. Out of fifty (8.2%) rodent specimens positive for Leishmania kDNA-PCR and sequencing, 10 were subsequently identified by sequencing of the ITS1 showing that five belonged to the L. donovani complex and five to L. tropica. Forty nine kDNA-positive rodents were found in the endemic localities of southern and eastern Ethiopia while only one was identified from northwestern Ethiopia. Moreover, all the ten ITS1-positive rodents were captured in areas where human leishmaniasis cases have been reported and potential sand fly vectors occur. Our findings suggest the eco-epidemiological importance of rodents in these foci of leishmaniasis and indicate that rodents are likely to play a role in the transmission of leishmaniasis in Ethiopia, possibly as reservoir hosts.     

First human cases of Leishmania (Viannia) naiffi infection in Ecuador and identification of its suspected vector species

Epidemiological surveillance of leishmaniasis was conducted in a northern Amazonian region of Ecuador, in which cutaneous leishmaniasis cases were recently reported. Sand flies were captured in the military training camp, and the natural infection of sand flies by Leishmania species was examined. Out of 334 female sand flies dissected, the natural infection by flagellates was microscopically detected in 3.9% of Lutzomyia yuilli yuilli and 3.7% of Lutzomyia tortura, and the parasite species were identified as Endotrypanum and Leishmania (Viannia) naiffi, respectively. After the sand fly surveillance, specimens from cutaneous leishmaniasis (CL) patients considered to have acquired the infection in the training camp area were obtained, and the infected parasite species were identified as L. (V.) naiffi. The present study reported first cases of CL caused by L. (V.) naiffi infection in Ecuador. In addition, a high ratio of infection of Lu. tortura by L. (V.) naiffi in the same area strongly suggested that Lu. tortura is responsible for the transmission of L. (V.) naiffi in this area.     

Molecular diagnosis of canine visceral leishmaniasis: Identification of Leishmania species by PCR-RFLP and quantification of parasite DNA by real-time PCR

The efficacies of polymerase chain reaction (PCR) procedures for the diagnosis of canine visceral leishmaniasis (CVL), and of PCR-restriction fragment length polymorphism (RFLP) analysis for the identification of Leishmania species, have been assessed. Quantitative real-time PCR employing a SYBR Green dye-based system was standardised for the quantification of Leishmania kDNA minicircles. Skin, peripheral blood and bone marrow samples collected from 217 dogs, asymptomatic or symptomatic for CVL, were analysed. The PCR method, which was based on the amplification of a 120 bp kDNA fragment conserved across Leishmania species, was able to detect the presence in clinical samples of protozoan parasite DNA in amounts as low as 0.1 fg. Bone marrow and skin samples proved to be more suitable than peripheral blood for the detection of Leishmania by PCR and presented positive indices of 84.9% and 80.2%, respectively. PCR-RFLP analysis indicated that 192 of the PCR-positive dogs were infected with Leishmaniainfantum chagasi, whilst L. braziliensis was identified in two other animals. Quantitative PCR revealed that bone marrow samples from dogs presenting positive conventional tests contained a higher number of copies of Leishmania kDNA than peripheral blood, although no significant differences were detected between symptomatic and asymptomatic dogs in terms of parasite load. This study demonstrates that PCR can be used for the detection of Leishmania in clinical samples derived from naturally infected dogs, and that PCR-RFLP represents a rapid and sensitive tool for the identification of Leishmania species. Additionally, qPCR is effective in quantifying Leishmania DNA load in clinical samples.     

Cutaneous leishmaniasis caused by Leishmania (L.) major infection in Sindh province, Pakistan

Leishmaniasis is endemic in Pakistan and is wide-spread throughout the country. Polymerase chain reaction (PCR) was performed to identify the Leishmania species present in cutaneous leishmaniasis (CL) patients from new endemic areas of the central part of Sindh province, Pakistan. The PCR primers used were designed for the identification and differentiation of Leishmania (Leishmania) major and Leishmania (Leishmania) tropica species, and PCR bands at 620 and 830 bp of the parasite-specific kinetoplast DNA sequences was identified for L. (L.) major and L. (L.) tropica, respectively. Among a total of 144 DNA samples purified from the skin biopsies of clinically suspected CL patients, 108 (75%) were positive for PCR amplification. Out of the 108 cases, 105 (97.2%) were determined to be positive for L. (L.) major infection, and 3 (2.8%) were positive for L. (L.) tropica infection. It was concluded that CL caused by L. (L.) major is the main source of infection in the central part of Sindh province in Pakistan. This rapid screening technique could be used for the diagnosis of a large number of samples from skin lesions, which commonly contain other bacterial and fungal infections.     

Sensitivity and reproducibility of a PCR assay for Leishmania detection using skin biopsy imprints on filter paper

The sensitivity and reproducibility of a PCR targeted to amplify the conserved 120 base-pair region of minicircles from Leishmania kDNA was defined using DNA extracted from skin biopsy imprints on filter paper. Seventy-seven patients with cutaneous leishmaniasis from an endemic region of Leishmania (Viannia) braziliensis in Brazil underwent skin biopsy of the ulcer border. Tissue samples were imprinted on filter paper and then, they were stored at −20 °C. Imprints on filter paper were stored at 4 °C. Samples were processed at three laboratories; Lab1 and Lab2 performed the PCR-kDNA assay using DNA extracted from the filter paper, and Lab3 processed PCR-kDNA using DNA from fresh-frozen tissue used as a gold standard. All samples were codified to maintain blinding during lab processing. Fifty-three (68.8%) patients had parasites isolated and identified by isoenzymes as L. (V.) braziliensis. The positivity of PCR-kDNA was similar between the three laboratories: 87.0, 85.7 and 88.3% (Lab1, Lab2 and Lab3, respectively). The sensitivity of PCR-kDNA in culture-proven cases was better, and showed similar results in all laboratories: 95.8, 95.8 and 97.9% (Lab1, Lab2 and Lab3, respectively). Data from the 77 enrolled patients showed an overall percent agreement of 80.5% (Kappa = 0.173) for the filter-paper approach between Lab1 and Lab2. Percent agreement between Lab1 and Lab3 was 83.1% (Kappa = 0.22), and it was 94.8% between Lab2 and Lab3 (Kappa = 0.77). Fifteen patients were diagnosed in just one of the two laboratories that used DNA extracted from filter paper. We conclude that the sensitivity of the filter paper approach is satisfactory and could be used in clinical trials and field work. Reproducibility could be improved using two separate imprints from the same biopsy sample.     

High infection frequency,low diversity of Leishmania major and first detection of Leishmania turanica in human in northern Iran

Smears of suspected patients infected with zoonotic cutaneous leishmaniasis (ZCL) were stained and examined under a light microscopic observation. DNA of parasites within human ulcers was extracted directly from their smears. Nested PCR was used to amplify a fragment containing the internal transcribed spacers of the ribosomal RNA genes (ITS-rDNA) of Lesihmania parasites in human from Turkemen Sahara located in the northeastern part of Iran. Based on RFLP method by digesting BsuRI restriction enzyme and more precisely sequencing of DNA ITS-rDNA was shown to be species-specific. The infection rates of Leishmania parasites were high with 154 (93.9%) infections out of 164 suspected patients using microscopic observations. Only from 128 suspected patients out of 164, ITS-rDNA fragments were amplified and 125 samples had enough DNA to digest BsuRI restriction enzyme and do DNA sequencing. The Nested PCR assays detected not only Leishmania major but also Leishmania turanica for the first time, another parasite of the great gerbil in human. The density of L. major was high but the diversity was low with only 2 haplotypes. The overall ratio of L. major (123 infections) to L. turanica (2 infections) was significantly higher (Chi-squared test: p < 0.05). Infections of L. turanica are not reported only and/or not known to cause human disease. Our analytical framework conveys a clear understanding of both L. major and L. turanica which can only be approved as causative agents of ZCL by more extensive sampling and followed by standardized molecular diagnosis.     

Combinations of ascaridole,carvacrol, and caryophyllene oxide against Leishmania

To date there are no vaccines against Leishmania and chemotherapy remains the mainstay for the control of leishmaniasis. The drugs currently used for leishmaniasis therapy are significantly toxic, expensive, and result in a growing frequency of refractory infections. In this study, we evaluated the effect of combinations of the main components of essential oil from Chenopodium ambrosioides (ascaridole, carvacrol, and caryophyllene oxide) against Leishmaniaamazonensis. Anti-leishmanial effects of combinations of pure compounds were evaluated in vitro and the fractional inhibitory concentration (FIC) indices were calculated. BALB/c mice infected with L. amazonensis were treated with different concentrations of ascaridole–carvacrol combinations by intralesional doses every 4 days. Disease progression and parasite burden in infected tissues were determined. In vitro experiments showed a synergistic effect of the combination of ascaridole–carvacrol against promastigotes of Leishmania with a FIC index of 0.171, while indifferent activities were observed for ascaridole–caryophyllene oxide (FIC index = 3.613) and carvacrol–caryophyllene oxide (FIC index = 2.356) combinations. The fixed ratio method showed that a 1:4 ascaridole–carvacrol ratio produced a better anti-protozoal activity on promastigotes, lower cytotoxicity, and synergistic activity on intracellular amastigotes (FIC index = 0.416). Significant differences (p < 0.05) in lesion size and parasite burden were demonstrated in BALB/c mice experimentally infected and treated with the ascaridole–carvacrol combinations compared with control animals. Carvacrol showed significant higher anti-radical activity in the DPPH assay compared with caryophyllene oxide. Electron spin resonance spectroscopy in combination with spin trapping suggested the presence of carbon-centered radicals after activation of ascaridole by Fe²⁺. The intensity of the signals is preferably decreased upon addition of carvacrol. The ascaridole–carvacrol combination could represent a future alternative to monotherapeutic anti-leishmanial agents.     

Amplified fragment length polymorphism (AFLP) analysis is useful for distinguishing Leishmania species of visceral and cutaneous forms

The Leishmania strains belonging to cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) have been reported to possess close homology in genome profiles. To confirm this on genetic basis an attempt was made to differentiate Leishmania major; Leishmania tropica and Leishmania donovani genetically for the first time using amplified fragment length polymorphism (AFLP)—a high throughput DNA fingerprinting technique. The objective of this research work was to identify DNA markers of CL and VL. Ten combinations of selective primers detect a total of 1487 informative AFLP marker. Percentage of polymorphism was 45.12%. Three hundred and thirty-seven unique AFLP markers were also identified in three species of Leishmania. A clear distinction was revealed between L. major and L. donovani. It was inferred by AFLP analysis that a higher rate of polymorphisms occurred among Leishmania species which indicate the distinguished pattern of the disease cause by Leishmania, i.e. VL and CL. Analysis based on polymorphic AFLP markers revealed considerably high genetic variation among the genome of these species which was sufficient to distinguish between CL and VL.     

In vivo activity of perifosine against Leishmania amazonensis

Miltefosine has been established as the first oral administration drug against cutaneous and visceral leishmaniasis. Other alkyl-phospholipids such as edelfosine have been tested against Leishmania showing an in vitro antiparasitic activity. Perifosine in vitro activity has been previously demonstrated against different Leishmania species including Leishmania amazonensis. In this study edelfosine and perifosine were orally administered to BALB/c mice at doses of 1 and 2.5 mg/kg/day during 28 days and 5 mg/kg/day during 14 days, starting the treatment 2 weeks after the first treatment scheme. Lesion sizes and parasitic burden as well as viability were determined in order to establish the treatment effectiveness. An assay to compare miltefosine at standard dose of 2.5 mg/kg/day during 28 days to an in vivo treatment with perifosine at the most effective treatment scheme observed in this study 5 mg/kg/day during 14 days, was also developed. Perifosine showed the higher activity in the in vivo assay and is showing as a new possibility within the alkyl-phospholipids group for the treatment against cutaneous leishmaniasis caused by L. amazonensis.     

Blood meal identification and parasite detection in laboratory-fed and field-captured Lutzomyia longipalpis by PCR using FTA databasing paper

The phlebotomine sand fly Lutzomyia longipalpis takes blood from a variety of wild and domestic animals and transmits Leishmania (Leishmania) infantum chagasi, etiological agent of American visceral leishmaniasis. Blood meal identification in sand flies has depended largely on serological methods but a new protocol described here uses filter-based technology to stabilise and store blood meal DNA, allowing subsequent PCR identification of blood meal sources, as well as parasite detection, in blood-fed sand flies. This technique revealed that 53.6% of field-collected sand flies captured in the back yards of houses in Teresina (Brazil) had fed on chickens. The potential applications of this technique in epidemiological studies and strategic planning for leishmaniasis control programmes are discussed.     

Leishmania mexicana lipophosphoglycan differentially regulates PKCα‐induced oxidative burst in macrophages of BALB/c and C57BL/6 mice

Leishmania are protozoan parasites that infect macrophages and their survival is partially achieved through inhibition of the cellular oxidative burst by parasite lipophosphoglycan (LPG). PKCα is the predominant PKC isoenzyme required for macrophage oxidative burst, yet it is not known if different susceptibility of BALB/c and C57BL/6 mice to Leishmania mexicana could be related to PKCα. We analysed the effect of L. mexicana promastigotes and parasite LPG on expression of PKCα and on its activity in macrophages of both mouse strains. Our data show that expression of the isoenzyme was not altered either by LPG or by L. mexicana promastigotes. Yet LPG exerted opposing effects on PKCα activity of macrophages between both strains: in susceptible BALB/c cells, it inhibited PKCα activity, whereas in the more resistant strain it augmented enzymatic activity 2·8 times. In addition, LPG inhibited oxidative burst only in susceptible BALB/c macrophages and the degree of inhibition correlated with parasite survival. Promastigotes also inhibited PKCα activity and oxidative burst in macrophages of BALB/c mice, whereas in C57BL/6, they enhanced PKCα activity and oxidative burst inhibition was less severe. Our data indicate that control of PKCα‐induced oxidative burst by L. mexicana LPG relates with its success to infect murine macrophages.     

Lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide via induction of oxidative stress in Leishmania (L.) infantum

Studying the cellular death pathways in Leishmania is an important aspect of discovering new antileishmanials. While using a drug repositioning approach, the lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide (NTZ) was investigated against Leishmania (L.) infantum. The in vitro antileishmanial activity and cytotoxicity were assessed using both parasite stages and mammalian NCTC cells, respectively. The lethal action of NTZ was investigated by detecting the phosphatidylserine (PS) exposure, reactive oxygen species (ROS) regulation, plasma membrane permeability, mitochondrial membrane potential and ultrastructural modifications by transmission electron microscopy. NTZ's activity against L. infantum was confirmed, producing IC₅₀ values of 42.71 μg/mL against promastigotes and 6.78 μg/mL against intracellular amastigotes. NTZ rapidly altered the cellular metabolism of promastigotes by depolarising the mitochondrial membrane and up-regulating the reactive oxygen species (ROS). In addition, the flow cytometry data revealed an intense and time-dependent exposure of PS in promastigotes. When using SYTOX^® Green as a fluorescent probe, NTZ demonstrated no interference in plasma membrane permeability. The ultrastructural alterations in promastigotes were time-dependent and caused chromatin condensation, plasma membrane blebbing and mitochondrial swelling. These data suggest that NTZ induced oxidative stress in L. (L.) infantum and might be a useful compound for investigating new therapeutic targets.     

Development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for Leishmania infection

Entomological monitoring of Leishmania infection in leishmaniasis endemic areas offers epidemiologic advantages for predicting the risk and expansion of the disease, as well as evaluation of the effectiveness of control programs. In this study, we developed a highly sensitive loop-mediated isothermal amplification (LAMP) method for the mass screening of sand flies for Leishmania infection based on the 18S rRNA gene. The LAMP technique could detect 0.01 parasites, which was more sensitive than classical PCR. The method was robust and could amplify the target DNA within 1 h from a crude sand fly template without DNA purification. Amplicon detection could be accomplished by the newly developed colorimetric malachite green (MG)—mediated naked eye visualization. Pre-addition of MG to the LAMP reaction solution did not inhibit amplification efficiency. The field applicability of the colorimetric MG-based LAMP assay was demonstrated with 397 field-caught samples from the endemic areas of Ecuador and eight positive sand flies were detected. The robustness, superior sensitivity, and ability to produce better visual discriminatory reaction products than existing LAMP fluorescence and turbidity assays indicated the field potential usefulness of this new method for surveillance and epidemiological studies of leishmaniasis in developing countries.     

Identification of Tunisian Leishmania spp. by PCR amplification of cysteine proteinase B (cpb) genes and phylogenetic analysis

Discrimination of the Old World Leishmania parasites is important for diagnosis and epidemiological studies of leishmaniasis. We have developed PCR assays that allow the discrimination between Leishmania major, Leishmania tropica and Leishmania infantum Tunisian species. The identification was performed by a simple PCR targeting cysteine protease B (cpb) gene copies. These PCR can be a routine molecular biology tools for discrimination of Leishmania spp. from different geographical origins and different clinical forms. Our assays can be an informative source for cpb gene studying concerning drug, diagnostics and vaccine research.     

Canine visceral leishmaniasis: Asymptomatic infected dogs as a source of L. infantum infection

Clinically infected dogs have been identified as the main reservoir hosts of visceral leishmaniasis (VL) caused by Leishmania infantum in the Mediterranean region. The objective of this study was to determine the potential of asymptomatic infected dogs compared with symptomatic ones as a source of L. infantum infection to golden hamster.For this purpose, anti-Leishmania antibodies were detected with direct agglutination test (DAT) in 13 symptomatic (7 seropositive = ≥1:320) and 53 asymptomatic (9 seropositive = ≥1:320 and 44 seronegative =  <1:320) ownership dogs. DNA of Leishmania sp. was extracted from skin and peripheral blood tissues of each dog and tested by PCR. Sixty-six Syrian golden hamsters (Mesocricetus auratus) were used for the determination of infectivity and pathogenicity of L. infantum, isolated from the dogs. We used the internal transcribed spacer 2 (ITS 2) rDNA sequence analysis. The results showed that 22 and 11 out of 66 inoculated golden hamsters were positive by PCR and parasitological examinations, respectively. From 22 PCR positive hamsters, 17 were related to asymptomatic dogs and 5 were from symptomatic ones. There was no significant difference between symptomatic and asymptomatic dogs in producing Leishmania infection in the susceptible animal model (P = 0.66). Smears and cultures of 5 dogs from 13 symptomatic dogs (38.5%) and 6 dogs from 53 asymptomatic ones (11.3%) were found to be positive at parasitological examination.All the L. infantum isolates from symptomatic and asymptomatic dogs were similar in sequencing.In conclusion, asymptomatic infected dogs as well as symptomatic ones can harbor L. infantum in their blood and skins which are virulent and infectious for inoculated golden hamster.     

Combination therapy with nitazoxanide and amphotericin B,Glucantime, miltefosine and sitamaquine against Leishmania (Leishmania) infantum intracellular amastigotes

Leishmaniasis is a neglected disease that affects poorest population mainly in developing countries, representing one of the major causes of mortality and morbidity. Therefore, efforts to find new chemotherapeutics for leishmaniasis remain a priority. Previous reports demonstrated the in vitro and in vivo antileishmanial activity of nitazoxanide, an antiprotozoan agent used in the treatment of infectious diarrhea. The present work was carried out to determine the effect of nitazoxanide in combination with current antileishmanial drugs. Mouse peritoneal macrophages were infected with Leishmania (Leishmania) infantum amastigotes in order to calculate the 50% and 90% inhibitory concentration values. Drug interactions were assessed with fixed ratio isobologram method and fractional inhibitory concentrations (FIC₅₀ and FIC₉₀); sum of FIC (ΣFIC₅₀ and ΣFIC₉₀) and overall mean ΣFIC (xΣFIC₅₀ and xΣFIC₉₀) were calculated for each combination. The nature of interactions was classified according to the xΣFIC₅₀ and xΣFIC₉₀. The combination between nitazoxanide and amphotericin B, Glucantime^®, miltefosine and sitamaquine showed xΣFIC₅₀ values of 1.13, 0.83, 1.06 and 0.94, respectively, indicating additive interaction. Considering the in vitro activity of nitazoxanide and the obtained results, further in vivo studies may be considered to evaluate possible drug interactions in visceral leishmaniasis.     

First report of natural infection in hedgehogs with Leishmania major, a possible reservoir of zoonotic cutaneous leishmaniasis in Algeria

We report here the first known cases of natural infection of hedgehogs with Leishmania major. Cutaneous leishmaniasis is an important public health problem in the area of M'sila, a semi-arid province in Algeria's northern Sahara, where two species of hedgehog live, Atelerix algirus and Paraechinus aethiopicus. The aim of this research was to survey Leishmania infection in these hedgehogs and evaluate whether they were reservoir hosts of Leishmania in an endemic zoonotic focus of leishmaniasis. Serological and molecular methods were used to determine the presence of Leishmania in 24 hedgehogs caught directly by hand and identified at species level as 19 A. algirus and 5 P. aethiopicus. Specific anti-Leishmania antibodies were detected in 29.2% of individuals by Western blot and in 26.3% by ELISA. The real-time PCR performed in spleen, ear and blood samples detected Leishmania spp. DNA in 12.5% of the individuals, one A. algirus and two P. aethiopicus. Three skin and two spleen samples of these animals were found to be parasitized and were identified by molecular test as L. major. Considering our results, it is suggested that hedgehogs have a potential epidemiological role as reservoir hosts of L. major.     

The influence of ecto-nucleotidases on Leishmania amazonensis infection and immune response in C57B/6 mice

Previous results from our laboratory and from the literature have implicated the expression of ecto-nucleotidases in the establishment of Leishmania infection. In the present study we evaluated the correlation between ecto-nucleotidasic activity and the infectivity of L. amazonensis promastigotes that were kept in culture for short or extended numbers of passages, a condition that is known to decrease parasite infectivity. We also analyzed the immune response associated with the infection by these parasites. As expected, we found that long-term cultured parasites induce the development of smaller lesions than the short-term cultured counterparts. Interestingly, long-term cultured parasites presented reduced ecto-nucleotidasic activity. In addition, cells recovered from animals infected with long-term cultured parasites produced higher amounts of IFN-γ and have smaller parasite load, after 8 weeks of infection. Furthermore, after 1 week of infection, there is increased expression of the chemokine CCL2 mRNA in animals infected with short-term cultured parasites. Finally, infection of peritoneal macrophages by these parasites also shows marked differences. Thus, while short-term cultured parasites are able to infect a greater proportion of macrophages, cells infected by long-term cultured parasites express higher amounts of CXCL10 mRNA, which may activate these cells to kill the parasites. We suggest that the enzymes involved in metabolism of extracellular nucleotides may have an important role in infection by L. amazonensis, by acting directly in its adhesion to target cells and by modulating host cell chemokine production.     

Immunological regulation of experimental cutaneous leishmaniasis. 1. Immunogenetic aspects of susceptibility to Leishmania tvopica in mice

Summary Models of the different disease patterns of cutaneous leishmaniasis can be induced by the same dose of L. tropica promastigotes in various inbred strains of mice. The susceptibility of BALB/c is exceptional, essentially dosage independent (being demonstrable with as few as 20 parasites) and leads to huge progressive lesions with fatal visceral and cutaneous metastasis. Lesions also extend progressively but more slowly in DBA/1 and DBA/2 mice. Strains A, C57BL/6 and CBA are relatively resistant to even 2 × 10⁷ promastigotes, with arrest of lesion growth within 3 weeks and subsequent gradual healing. Similar resistance of A.SW to 2 × 10⁵ is overcome by a larger dose. The major inter-strain differences are H-2 independent, for C57BL/10 congenic mice possessing six different H-2 antigen complexes all show early arrest of lesion growth leading to healing (H-2^s H-2^a, H-2^k) or mild residual disease (H-2^b H-2^d, H-2^q). Inter-line differences within the latter group varied between experiments such that no clear rank order emerged. Inexorable disease progression was found in congenic BALB/B, BALB/c and BALB/K alike, although it was significantly slower in the latter line when infected with smaller doses. Genetic control of BALB/c susceptibility is thus predominantly in the non-H-2 background with only a minor H-2 linked regulatory influence on the later stage. C57BL/6, BALB/c and their F1 hybrid characteristically display ‘healing’, ‘fatal progressive’ and ‘non-healing’ lesions respectively over a wide dose range. ‘BALB/c-like’ susceptibility segregates strictly in the F2 and backcross progeny according to a one predominant gene prediction. A comparison of the present data with those concerning genetic regulation of acute and chronic stages of systemic L. donovani infection in mice (Bradley, 1977, Blackwell, Freeman & Bradley 1980) reveals differing control for the outcome of cutaneous L. tropica infection, in which other important genetic influences must be involved.