Genetic analysis of Leishmania mexicana populations from Texas, Latin America, and the Caribbean.

A genetic analysis using enzyme data of 72 Leishmania mexicana isolated from hosts in Texas, Latin America, and the Caribbean is presented. All isolates from each country were combined and considered as local populations. Allomorph (allele determined by electrophoresis) frequencies for 20 enzyme (loci) were calculated and 7 populations (Texas, Mexico, Belize, Guatemala, Ecuador [EC], Venezuela, and the Dominican Republic [DR]) were compared pairwise in the statistic of genetic identity (I) (level of genetic similarity). All populations were found to be genetically similar with a mean I value for all comparisons of 0.890 +/- 0.067. When DR was included as one of the pair compared, I = 0.811 +/- 0.034. Among comparisons that include EC (excluding EC vs. DR), I = 0.875 +/- 0.026. The mean I for the other comparisons was less than 0.9. The data indicate that the DR population is divergent enough from the others that it can be considered at the subspecies/incipient species level of evolutionary divergence; the EC population is, to a lesser extent, distinct from the others, and the other 5 represent geographic populations of 1 widely distributed species. A diagrammatic representation of the allomorphs among the 72 isolates is included. There were some allomorph/geographical (or local) population relationships noted.

Genetic similarity among Central and South American populations of Leishmania (Viannia) braziliensis.

Four populations of Leishmania (Viannia) braziliensis from Central America, Colombia, Peru and Brazil were analyzed and compared for up to 20 enzyme loci. Each of the 180 isolates could be identified as L. braziliensis using combined data from glucose phosphate isomerase and mannose phosphate isomerase. When the most common enzyme band was present at a frequency of < or = 0.95, the populations were polymorphic (more than a single allomorph for an enzyme) for more than 50% of the loci. Included were diagrammatic representations of the enzyme polymorphisms. Comparisons of levels of enzyme polymorphism and of genetic similarity among other Leishmania populations, L. tropica, L. major, L. mexicana, and L. donovani sensu lato, were discussed. The mean +/- SD level of genetic similarity among the four populations was 0.924 +/- 0.036 (range 0.878-0.966), which indicates that L. braziliensis is probably one reproductive population from Mexico in the north to Brazil and Peru in the south.

Biochemical characterization of Trypanosoma spp by isozyme electrophoresis.

Isozyme patterns of 13 enzymes were compared for cultures of Trypanosoma avium, T. vespertilionis, T cruzi and T. rangeli. The isozyme separation was made by cellulose acetate electrophoresis. Each of the species had distinctly migrating isozyme bands for glutamate oxaloacetate transaminase (GOT), isocitrate dehydrogenase (ICD), malic enzyme (ME), 6-phosphogluconate dehydrogenase (6PGD), phosphoglucoisomerase (PGI), phosphoglucomutase (PGM), and malic dehydrogenase (MDH). For other enzymes, two or more species had identically migrating bands. In addition to these interspecific species differences, variability was observed among the strains of T. cruzi and T. rangeli. Among the T. cruzi strains, there were two different isozyme (possibly allozyme) types of the enzymes alanine aminotransferase (ALAT), fructokinase (FK), glucose-6-phosphate dehydrogenase (G6PDH), GOT, MDH and three types of ME. In the T. rangeli isolates two isozyme types for the enzymes ALAT, FK, G6PDH, GOT, ICD, and LDH, were observed. Among the eight strains of T. cruzi studied there were six isozyme types, and among the seven T. rangeli isolates there were four isozyme types. There was an indication that isozyme types were associated with geographical distribution.

Characterization of Leishmania spp. by isozyme electrophoresis.

In this study, isozyme patterns for 14 different enzymes were compared for culture strains of Leishmania braziliensis, L. hertigi, L. mexicana, L. donovani, L. tropica, and L. adleri. The isozyme separation was made by means of cellulose acetate electrophoresis. Each of the species had distinct isozyme patterns for aspartate aminotransferase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and fructokinase. For other enzymes, two or more species had identically migrating bands; however, by using combinations of the other 10 enzymes it was possible to separate any one of the six species. In addition to these interspecific differences the Panama strains of L. braziliensis had two different malic dehydrogenase isozyme patterns; therefore, they fell into two distinct groups. These strains otherwise had identical isozyme patterns.

Biochemical identities and differences among Leishmania species and subspecies.

An analysis was presented for identification of 20 species and subspecies of Leishmania by cellulose acetate electrophoresis data from the enzymes glucose phosphate isomerase, mannose phosphate isomerase, and phosphogluconate dehydrogenase. Most Leishmania could be identified from data of these three enzymes. The CAE data for 20 enzymes from over 300 New and Old World isolates were combined, and an analysis of the data which included calculations of genetic identities and genetic distances was reported. High levels of genetic similarity and low levels of genetic distance were noted among comparisons of local populations of the same Leishmania, and lower levels of similarity and higher levels of distance were noted among intracomplex pairings. The biochemical data suggested that similarities and differences among Leishmania could be quantified as they have been in other organisms. For the most part the data presented were consistent with the taxonomic rankings which were based on morphology, behavior, ecology, and other biochemical data.

Identification of Leishmania spp. by multiple isozyme analysis.

Biochemical data as enzyme profiles which were obtained by cellulose acetate electrophoresis (CAE) are reported on 44 Leishmania isolates. These enzyme profiles contain data from 25 enzyme systems. Calculations from the CAE data on average polymorphism indicated that Leishmania species/types or groups can be expected to be about 25% polymorphic, which suggests that isolate pairs which have profiles about 75% or more identical should be considered samples from the same species/type, and isolates that are significantly less than 75% identical are therefore samples from different species/types. There were five major groupings of isolates according to enzyme profiles, which were for the most part consistent with groupings of the genus based on other criteria: braziliensis, mexicana, donovani, tropica and hertigi profiles. Within these groups there were natural subgroups of isolates among which there was 75% or more allozyme or allomorph (genetic) identity. The braziliensis profile group had two subgroups: panamensis and braziliensis or guayanensis, and the mexicana profile group had three subgroups: mexicana, amazonensis and peruviana. There was an indication that an L. d. infantum isolate might be different from the other L. donovani isolates, and that the L. tropica isolates could be samples from more than one group. The data reported here are consistent with previously reported CAE data, but suggest that isozyme analysis of Leishmania isolates leading to identification should be based on data from many enzyme systems rather than just a few.

Characterization of a Leishmania isolate from the rodent host Neotoma micropus collected in Texas and comparison with human isolates.

We report the biological and biochemical parameters of Leishmania parasites (MNEO/US/90/WR972) isolated from a rodent host, Neotoma micropus, collected in Texas. Footpad inoculations of WR972 promastigotes into BALB/c mice and Syrian hamsters resulted in ulcerating lesions six and eight weeks post-inoculation, respectively. Using monoclonal antibody-stained touch preparations, amastigotes were found in the liver of both laboratory hosts. Infection of J774 macrophages with WR972 promastigotes supported the growth of amastigotes for 12 days at 35 degrees C. The WR972 parasite was identified by enzyme electrophoresis as L. mexicana. Isozyme comparison of WR972 with 42 L. mexicana isolates (from humans and rodents) from four different endemic areas, including Texas, suggest that these parasite populations are identical for approximately 97% of their genetic loci. Pulse field gel electrophoresis (PFGE) of WR972 resolved 18 chromosomes with a size range of 300- greater than 2,000 kb. The karyotype strongly resembles that of two other Texas L. mexicana isolates from humans. Taken together, the PFGE, hybridization, and isoenzyme data suggest that the wood rat isolate (WR972) is identical to parasites from human cutaneous lesions isolated in Texas and Central America. In addition, the biological characteristics of WR972, its infectivity of BALB/c mice and the Syrian hamster, and the potential of the isolate to infect, transform, and divide in J774 macrophages indicate that WR972 will be pathogenic in humans if transmission occurs. Health care providers should consider this possibility when studying the epidemiology and control of cutaneous leishmaniasis in Texas.

Leishmania colombiensis in Venezuela.

Flagellate parasites isolated in Venezuela from bone marrow aspirates of a human (MHOM/VE/70/Chuao) and a dog (MCAN/VE/72/Talisman2) were subsequently identified by isozyme analysis as Leishmania colombiensis. Data are presented describing genetic similarity among Panama, Colombia, and Venezuela populations of this species.

Identification of Leishmania isolates from a Lebanese population.

We report the results of enzymatic patterns of isolates of Leishmania cultured from patients referred to Department of Dermatology of the American University of Beirut Medical Center in Lebanon. The results reveal that these Lebanese isolates are all very similar despite variable clinical presentations of the patients and differences in geographic origin. Leishmania donovani sensu lato is the dominant species present in the skin lesions observed; thus, clinical manifestations and/or geographic distribution cannot be used as reliable criteria for identifying Leishmania parasites from this geographic area. Enzyme data should be combined with these parameters before definitive identification can be made.

Leishmania (Viannia) lainsoni: first isolation in Peru.

Surveys were conducted from 1986 through 1992 to define the etiology and geographic distribution of human leishmaniasis in Peru. Lesion aspirates and skin biopsies were obtained from clinically diagnosed cases of leishmaniasis and tested for promastigotes by standard culture techniques. The isozyme profile of the isolates was determined by the cellulose acetate electrophoresis technique. Data indicated that the isozyme profiles for Leishmania isolates from six patients were similar to that of reference strains of L. lainsoni. These results are the first reported evidence of L. lainsoni and the first association of this parasite with human cases of cutaneous leishmaniasis in Peru.

Evidence of sexual reproduction in the protozoan parasite Leishmania (Kinetoplastida: Trypanosomatidae).

Parasitic protozoa of the genus Leishmania (Kinetoplastida: Trypanosomatidae) are generally thought to multiply by binary fission; however, data from quantitative microspectrophotometry indicate that nuclear fusion or sexual reproduction takes place in the intracellular amastigote form. Among several different Leishmania species, the mean +/- SD nuclear DNA content of all promastigotes (extracellular form) and of some amastigotes (intracellular form) in macrophages was 0.098 +/- 0.032 relative units; in contrast, other amastigotes within the same macrophage had a mean +/- SD nuclear DNA content of 0.219 +/- 0.050. The latter population of amastigotes are apparently produced when the nuclei of a pair of 0.098 amastigotes fuse. These 0.219 amastigotes later go through what is probably the typical meiotic cycle to reform the 0.098 condition we observed among promastigotes. The demonstration of sexual reproduction in Leishmania has important implications for the future direction of research on this medically important parasite.

Identification, using isoenzyme electrophoresis and monoclonal antibodies, of Leishmania isolated from humans and wild animals of Ecuador.

Six strains of Leishmania isolated from wild mammals and humans on the Pacific Coast of Ecuador were identified by isoenzyme electrophoresis and by their reactivity patterns to a cross-panel of specific monoclonal antibodies using a radioimmune binding assay. Single isolates from Sciurus vulgaris, Potos flavus, and Tamandua tetradactyla were identified as Leishmania amazonensis. Three other strains, isolated from cutaneous lesions of humans, were identified as Leishmania panamensis.

Human cutaneous leishmaniasis in Ecuador: identification of parasites by enzyme electrophoresis.

Twenty-six strains of Leishmania were isolated from cutaneous lesions in humans in 3 different geographical areas of Ecuador. The species were identified by enzyme electrophoresis as Leishmania braziliensis, L. panamensis, L. guyanensis, L. mexicana, and L. amazonensis.

Distribution and etiology of leishmaniasis in Colombia.

A total of 340 Leishmania strains, isolated from humans, animals, and sand flies from various regions of Colombia, were examined by isozyme electrophoresis. Seven different Leishmania species were identified. Leishmania panamensis and L. braziliensis were the most common, representing 53.8% and 30.3% of the total, respectively. Isolation rates of the other species were as follows: L. chagasi, 9.4%; L. guyanensis, 2.6%; L. amazonensis, 1.8%; L. mexicana, 0.8%; and a new species requiring additional study, 1.2%. Statistical analyses of representative L. panamensis and L. braziliensis isolates indicated that the populations of these 2 species are genetically very similar. L. panamensis may have a continuous distribution in Colombia west of the eastern Andes Mountains and L. braziliensis may have a continuous distribution east of the western Andes Mountains. Information is given on disease manifestations of the parasites in human hosts and on isolation records from sand flies and animals.

Characterization of Leishmania colombiensis sp. n (Kinetoplastida: Trypanosomatidae), a new parasite infecting humans, animals, and phlebotomine sand flies in Colombia and Panama.

Characterization of Leishmania colombiensis sp.n. is presented, which on the basis of biological and molecular criteria, appears to be a new member of the L. braziliensis complex. A total of nine isolates of the new parasite were made in Colombia and Panama between 1980 and 1986: two from human cases of cutaneous leishmaniasis, six from phlebotomine sand flies, and one from a sloth. Although most closely related to L. lainsoni, L. colombiensis sp.n. is clearly distinguishable from other members of the genus by its reactivity with monoclonal antibodies, isoenzyme electrophoresis, and restriction endonuclease fragment patterns of kinetoplast DNA (k-DNA).

Identification and genetic comparison of leishmanial parasites causing viscerotropic and cutaneous disease in soldiers returning from Operation Desert Storm.

Six Leishmania major and seven L. tropica parasites were isolated and identified from participants in Operation Desert Shield/Storm. A complete enzyme analysis (21 enzymes) revealed that there was enzyme polymorphism among the isolates of each species group. Any one Desert Storm L. major isolate could differ from any other for 1-3 enzymes, and any L. tropica isolate could differ from any one other for up to eight enzymes. Enzyme polymorphism data from other L. major and L. tropica isolates from Africa and the Middle East region were obtained and combined with the Desert Storm data to produce population enzyme polymorphism estimates. Results from these population data indicated that L. major parasites could be expected to differ from each other for as many as eight enzymes and still be L. major, and similarly, L. tropica isolates could differ for as many as 14 enzymes. These expected isolate variation extremes have not been observed among the isolates studied. All L. major and most L. tropica isolates were from patients who, as expected, presented with cutaneous disease, but the Desert Storm and two Kenyan patients infected with L. tropica presented with a viscerotropic disease, the symptoms of which are unlike those of classic visceral leishmaniasis. Such unrecognized presentation for these L. tropica-infected patients indicates that both parasite and patient can play critical roles in disease manifestations. The Desert Storm isolates are, as indicated, either L. major or L. tropica.

Epidemiology of cutaneous leishmaniasis at a focus monitored by the multinational force and observers in the northeastern Sinai Desert of Egypt.

A longitudinal epidemiologic study of cutaneous leishmaniasis (CL) transmission was conducted between July 1989 and June 1991 in a 1,200-km2 sector of the northeastern Sinai Desert monitored by the Multinational Force and Observers (MFO), an international peace keeping mission between Egypt and Israel. The occurrence of human cases, sand fly density, rodent collection, and isolations of Leishmania confirmed only one of four surveyed locations as a significant focus of CL transmission. Phlebotomus papatasi, the only anthropophilic sand fly species encountered at this focus, comprised more than 96% of the sand fly population and attained human landing densities exceeding 100 sand flies/person/hr during 1990. Seasonal activity of this species ranged from April to November, with highest densities occurring during the period May-September. A peak promastigote infection rate of 2.4% (13 of 534) was observed in P. papatasi during July 1990. Twelve of the 60 (20%) persons at risk during the six months of intense sand fly activity at this site developed lesions consistent with CL; L. major was isolated from nine (75%) of these cases. Leishmania major infection was acquired by two of 22 (9%) sentinel hamsters used during the same period. More than 97% of the 897 wild rodents trapped at this site were desert gerbil species. Leishmania major was the only Leishmania isolated from human, sand fly, wild rodent (Gerbillus pyramidum), and sentinel hamster infections that originated at site Check point 1-Delta, the focus of CL transmission within jurisdiction of the MFO. The altered ecology of this area, created by construction of a dam, may contribute significantly to the transmission dynamics of CL at this focus.

Epidemiology of visceral leishmaniasis in Colombia.

Epidemiologic studies were conducted during the period 1986-1988 in a small rural community in Colombia (El Callejon) where visceral leishmaniasis is highly endemic. In this community of 185 people, 14 cases of infantile visceral leishmaniasis were diagnosed in the 9 years 1981-1988. Leishmanin skin testing of a sample of the human residents showed that prevalence of Leishmania chagasi infection increased with age; overall, 51.2% of the subjects had a positive reaction. A canine surveillance program was instituted, using introduced sentinel dogs as well as the indigenous dog population. Eleven of 16 sentinel dogs were infected within 8 months of exposure; mean seroconversion time was 4.4 months. Eleven of 25 seronegative local dogs were also infected during the 26 month period; mean seroconversion time was 8 months. Parasites identified by isozyme electrophoresis as L. chagasi were recovered from 18 of 22 seropositive dogs. Collections of wild animals using baited live traps yielded mainly the neotropical opossum, Didelphis marsupialis. Leishmania chagasi was recovered from 12 of 37 (32.4%) opossums. Six of 681 female Lutzomyia longipalpis collected in the community had flagellates in their guts; cultures from 4 were identified as L. chagasi. These data confirmed that active parasite transmission occurred. The relatively high prevalence of L. chagasi infection found among D. marsupialis captured near human dwellings suggests that these animals may be an important peridomestic reservoir.

Geographic distribution and clinical description of leishmaniasis cases in Peru.

Studies were conducted from 1986 through 1993 to further define the geographic distribution and relative importance of different species of Leishmania as a cause of leishmaniasis in Peru. Patients with a clinical diagnosis of cutaneous and/or mucosal or diffuse cutaneous leishmaniasis were enrolled at the Naval Medical Research Institute Detachment (NAMRID) Laboratory in Lima, the Tropical Disease Clinic at San Marcos University Daniel A. Carrión, the Central Military Hospital, and a Ministry of Health hospital in Cusco, Peru. Clinical features, lesion aspirates, and biopsy tissue were obtained from each patient. All specimens were collected and assayed separately, including multiple specimens from some of the same patients for Leishmania parasites by inoculating aliquots of either aspirates or biopsy tissue suspensions onto Senekji's blood agar medium. Stocks of Leishmania isolates were used to prepare promastigotes to produce extracts for identifying the Leishmania species by the cellulose acetate electrophoresis enzyme technique. A total of 351 isolates of Leishmania were obtained from 350 patients who were infected primarily in the low and high jungle of at least 15 different Departments of Peru. Of the 351 isolates, 79% were identified as L. (V.) braziliensis, 7% as L. (V.) guyanensis, 10% as L. (V.) peruviana, 2% as L. (V.) lainsoni, and 1.7% as L. (L.) amazonensis. The clinical form of disease varied depending on the species of Leishmania, with L. (V.) braziliensis being associated most frequently with cutaneous, mucosal ulcers and mixed cutaneous and mucosal disease, and L. (V) peruviana, L. (V.) guyanensis, L. (V.) lainsoni with cutaneous lesions. Leishmania (L.) amazonensis was isolated from six patients, three with cutaneous lesions, one with mucosal lesions, and two with diffuse cutaneous lesions. Among all of the leishmaniasis cases, males were affected more frequently, and cases occurred among patients less than 10 to more than 51 years of age. These data further defined the geographic distribution and the relative frequency of Leishmania species associated with different clinical forms of leishmaniasis in Peru.

Isolation of Leishmania mexicana (Kinetoplastida: Trypanosomatidae) from Lutzomyia anthophora (Diptera: Psychodidae) collected in Texas.

Three of 27 female Lutzomyia anthophora (Addis) collected in Texas from the nest of a southern plains woodrat, Neotoma micropus Baird, during October 1991 were infected with flagellate protozoans. Isolates were grown in Schneider's Drosophila medium supplemented with 20% fetal bovine serum, and isozyme analysis of two of the isolates determined the parasites to be Leishmania mexicana (Biagi). These are the first isolations of Leishmania from field-collected sand flies in North America north of Mexico. Possible reasons for the lack of human cases near the focus are presented.