The ornithine decarboxylase gene of Trypanosoma brucei: Evidence for horizontal gene transfer from a vertebrate source.

Kinetoplastid protozoans in the family Trypanosomatidae are parasites, many of them responsible for serious diseases in humans and domestic animals. Ornithine decarboxlyase (ODC), a protein at the core of polyamine metabolism, is a potential target for therapies to overcome these diseases. Eukaryotic phylogenies were constructed from full-length genes for ODC to determine the origin of ODC in the kinetoplastid protozoans. The Odc genes from Trypanosoma brucei and two other African trypanosomes, T. congolense and T. vivax, clustered with Odc genes from vertebrates rather than with Odc genes from other kinetoplastids and other protozoans, making this gene a candidate for horizontal gene transfer from a vertebrate source. This result is unique to the Odc gene from the African trypanosomes as four other genes produced phylogenies consistent with the expected taxonomic relationships for the organisms. Analysis of the genomic regions around the Odc genes in Leishmania major, T. brucei, and Trypanosoma cruzi supports the hypothesis of loss of the Odc gene in the Trypanosoma lineage followed by acquisition of a new copy from a vertebrate host in the African branch of the genus.     

Direct identification of Leishmania species in biopsies from patients with American tegumentary leishmaniasis

Accurate identification of Leishmania species is important for monitoring clinical outcome, adequately targeting treatment, and evaluation of epidemiological risk in tegumentary leishmaniasis. This is especially the case in regions where several species coexist and for travel medicine where the geographical source of infection is not always obvious. Species identification presently depends on parasite isolation, which is not very sensitive and not necessarily representative of parasites actually present in human tissues. We evaluated a polymerase chain reaction (PCR) assay combining amplification of the gp63 genes and restriction fragment length polymorphism (RFLP) analysis (gp63 PCR-RFLP) for direct Leishmania species-identification in tissues collected from Peruvian patients in 1999. By comparison with a kinetoplast DNA-based PCR, our PCR assay showed a detection sensitivity of 85%. Three species were encountered among patient samples, Leishmania (Viannia) braziliensis, L. (V.) peruviana and L. (V.) guyanensis, and their frequency and geographical distribution corresponded to earlier epidemiological studies of leishmaniasis in Peru. However, unexpected results raised questions about (i) the contribution of human migration to the emergence of new foci of given species, (ii) the pathogenicity of some species, and (iii) the frequency of mixed or hybrid infections.     

Assessment of nuclear and mitochondrial genes in precise identification and analysis of genetic polymorphisms for the evaluation of Leishmania parasites

The polymorphism and genetic diversity of Leishmania genus has status under discussion depending on many items such as nuclear and/or mitochondrial genes, molecular tools, Leishmania species, geographical origin, condition of micro-environment of Leishmania parasites and isolation of Leishmania from clinical samples, reservoir host and vectors. The genetic variation of Leishmania species (L. major, L. tropica, L. tarentolae, L. mexicana, L. infantum) were analyzed and compared using mitochondrial (COII and Cyt b) and nuclear (nagt, ITS-rDNA and HSP70) genes. The role of each enzymatic (COII, Cyt b and nagt) or housekeeping (ITS-rDNA, HSP70) gene was employed for accurate identification of Leishmania parasites. After DNA extractions and amplifying of native, natural and reference strains of Leishmania parasites, polymerase chain reaction (PCR) products were sequenced and evaluation of genetic proximity and phylogenetic analysis were performed using MEGA6 and DnaSP5 software. Among the 72 sequences of the five genes, the number of polymorphic sites was significantly lower as compared to the monomorphic sites. Of the 72 sequences, 54 new haplotypes (five genes) of Leishmania species were submitted in GenBank (Access number: KU680818 – KU680871). Four genes had a remarkable number of informative sites (P = 0.00), except HSP70 maybe because of its microsatellite regions. The non-synonymous (dN) variants of nagt gene were more than that of other expression genes (47.4%). The synonymous (dS)/dN ratio in three expression genes showed a significant variation between five Leishmania species (P = 0.001). The highest and lowest levels of haplotype diversity were observed in L. tropica (81.35%) and L. major (28.38%) populations, respectively. Tajima's D index analyses showed that Cyt b gene in L. tropica species was significantly negative (Tajima's D = − 2.2, P < 0.01), while COII and nagt genes were produced through evolutionary processes for both L. tropica and L. major (Tajima's D = 2.85 & 2.91, P < 0.01). More different clinical lesions with extensive phylogenetic and evolutionary analyses should be employed to avoid confusion in the diagnosis of leishmaniasis and development of vaccines for eradicating Leishmania parasites.     

Synthesis of oxysterols and nitrogenous sterols with antileishmanial and trypanocidal activities

Two sterol families have been synthesized: the first one is nitrogenous sterols containing amino, N-hydroxyimino or cyano group and the second one is oxysterols such as ketosterol and hydroxysterols. These compounds were then evaluated in vitro against Leishmania donovani promastigotes and Trypanosoma brucei brucei trypomastigotes. The most active compounds against L. donovani promastigotes were 7beta-aminomethylcholesterol and 7alpha,beta-aminocholesterol (IC50 in a range from 1 to 3 microM, pentamidine: 2.8 microM). These compounds were active on intramacrophage amastigotes with IC50 of 1.3 microM. Such an activity justifies further in vivo antileishmanial evaluation. Against T. b. brucei, (24R,S)-24-hydroxy-24-methylcholesterol (MEC, 12.5 microM) was the most active compound from these series.     

Distribution of constitutive heterochromatin in two species of triatomines: Triatoma lenti Sherlock and Serafim (1967) and Triatoma sherlocki Papa,Jurberg, Carcavallo,Cerqueira & Barata (2002)

Triatoma lenti and Triatoma sherlocki are hemipterans that belong to the brasiliensis subcomplex. In triatomines, the constitutive heterochromatin pattern is species-specific and allows, in many cases, for the grouping of species. Thus, we cytogenetically analyzed T. sherlocki and T. lenti using C-banding, and we compared the results with previous ones obtained in other species of the brasiliensis subcomplex. Both species were found to have a male diploid chromosome number of 22 chromosomes (2n = 20A + XY) with heterochromatic blocks at one or both chromosomal ends of all autosomal pairs. During early meiotic prophase, they showed a large heteropycnotic chromocenter constituted by the association of both sex chromosomes plus two autosomal pairs and many heterochromatic blocks dispersed inside the nucleus. All of these cytogenetic characteristics are similar to those observed in other species of brasiliensis subcomplex, results which confirm the grouping of T. sherlocki and T. lenti within this subcomplex. However, we emphasize the importance of other approaches, such as molecular analysis, to confirm the placement of T. lenti within the brasiliensis subcomplex.     

Visceral leishmaniasis in India: promises and pitfalls of a PCR-based blood test

Traditional methods of diagnosing visceral leishmaniasis (kala-azar) in India suffer from a number of disadvantages. Amplification of multicopy nuclear genes and messenger ribonucleic acid of Leishmania by the polymerase chain reaction (PCR) was evaluated as an alternative assay under various clinical conditions. PCR of peripheral blood has the highest absolute sensitivity among all the available procedures, and is particularly useful for detecting parasites in early infections, post kala-azar dermal leishmaniasis, concurrent infections and immunocompromised cases, but is not so reliable for late infections. PCR of immunopurified blood mononuclear cells indicated the association of parasites with monocytes as well as non-monocyte cell types.     

American tegumentary leishmaniasis: antigen-gene polymorphism, taxonomy and clinical pleomorphism.

Multi-locus enzyme electrophoresis is the current gold standard for the genetic characterisation of Leishmania. However, this method is time-consuming and, more importantly, cannot be directly applied to parasites present in host tissue. PCR-based methods represent an ideal alternative but, to date, a multi-locus analysis has not been applied to the same sample. This has now been achieved with a sample of 55 neotropical isolates (Leishmania (Viannia) braziliensis, L. (V.) peruviana, L. (V.) guyanensis, L. (V.) lainsoni and L. (L.) amazonensis), using five different genes as targets, four of which encoded major Leishmania antigens (gp63, Hsp70, H2B and Cpb). Our multi-locus approach strongly supports the current taxonomy and demonstrates a highly robust method of distinguishing different strains. Within L. (V.) braziliensis, we did not encounter so far specific genetic differences between parasites isolated from cutaneous and mucosal lesions. Interestingly, results provided by each of the different antigen-genes in the species considered, were different, suggesting different selective pressures. Our work emphasises the need for a multi-disciplinary approach to study the clinical pleomorphism of leishmaniasis.     

Natural infections of Leishmania peruviana in animals in the Peruvian Andes.

Evidence that domestic dogs may act as reservoir hosts for cutaneous leishmaniasis in the Peruvian Andes is provided by the isolation, for the first time, from naturally infected dogs of parasites identified (by isoenzymes) as Leishmania peruviana. Leishmania parasites were isolated from nasal aspirates or biopsies from 5 (1.8%) of 279 asymptomatic dogs samples in endemic villages of the Peruvian Andes. In addition, Leishmania (Viannia) infections were identified in 15 (5.4%) of 276 nasal samples by the polymerase chain reaction (PCR) using subgenus-specific primers. Further circumstantial evidence for a reservoir role for dogs comes from the finding of a relatively high dog blood index among the sandfly vectors collected inside houses (29% for Lutzomyia peruensis and 17% for Lu. verrucarum). Possible wild mammal reservoir hosts for Andean cutaneous leishmaniasis were also detected in endemic villages. At least 8 species were identified among the 1266 small mammals trapped. Leishmania parasites were isolated from blood or skin biopsies taken from 2 (2.6%) of 78 Didelphis albiventris and 6 (1.2%) of 511 Phyllotis andinum. Three isolates were identified by isoenzymes as L. peruviana, and the other 5 were identified by PCR as Leishmania (Viannia) species. Leishmania (Viannia) infections were also identified by PCR directly on skin biopsies taken from 2 (2.8%) of 72 D. albiventris, 1 (0.2%) of 499 P. andinum, and 4 (2.6%) of 153 Akodon sp.     

The origin and evolution of the Leishmania donovani complex as inferred from a mitochondrial cytochrome oxidase II gene sequence.

Members of the Leishmania donovani complex are parasites of the reticulo-endothelial system that are often associated with serious epidemics of a life threatening disease known as visceral leishmaniasis or kala-azar. Twenty-two Leishmania isolates representative of the geographical range of the parasite were analysed for sequence variations in their cytochrome oxidase II gene. In performing phylogenetic analysis, the maximum parsimonious, neighbour joining and maximum likelihood trees were congruent and produced a tree that differentiated between two clades conforming to the current classification of the species complex into two species: Leishmania donovani and Leishmania infantum. Furthermore, the molecular haplotypes were concordant, in general, with the isoenzyme data of the complex. The donovani isolates from the Sudan that possessed the most ancestral sequence were of a single haplotype that significantly resembled the sequence of Leishmania major. Our sequence data tallied with a general neutral model of sequence evolution with manifestations of weak selection. The data allowed an approximate dating of the origin of the complex to a period contemporary to or predating the spread of modern humans out of Africa.     

Comparison of Three Commonly Used Genetic Markers for Detection of Leishmania Major: An Experimental Study

BackgroundLeishmaniasis is a vector-borne disease caused by an intracellular protozoan parasite called Leishmania spp. Different species produce different clinical outcomes; the majority of cases are cutaneous forms. Leishmania major is one of the main causative agents of cutaneous leishmaniasis (CL). Various methods are being using to diagnose CL, including microscopic examination, culture, and molecular detection of the parasite genome.MethodIn the current study, we tried to compare three common molecular markers, including Kinetoplast DNA (kDNA), Cytochrome b (Cyt b), and Internal transcribed space 1 (ITS1), for the detection of Leishmania major. After cultivation of standard strain of L. major MHOM/IR/75/ER in RPMI 1640, certain number of promastigotes was subjected to DNA extraction and different PCR reactions.ResultsThe lowest number of the parasite (5 promastigotes) can be detected by kDNA-PCR, followed by Cyt b-PCR (10 promastigotes), and ITS1-PCR (50 promastigotes).ConclusionIn conclusion, kDNA-PCR was the most sensitive marker and may provide more reliable data in the initial screening, especially in false-negative results provided by parasitological methods due to the low number of parasites.     

Further evidence from SSCP and ITS DNA sequencing support Trypanosoma evansi and Trypanosoma equiperdum as subspecies or even strains of Trypanosoma brucei

The subgenus Trypanozoon includes three species Trypanosoma brucei, Trypanosoma evansi and Trypanosoma equiperdum, which are morphologically identical and indistinguishable even using some molecular methods. In this study, PCR-based single strand conformation polymorphism (PCR-SSCP) was used to analyze the ribosomal DNA of the Trypanozoon species. Data indicate different patterns of ITS2 fragments between T. brucei, T. evansi and T. equiperdum by SSCP. Furthermore, analysis of total ITS sequences within these three members of the subgenus Trypanozoon showed a high degree of homology using phylogenetic analysis but were polyphyletic in haplotype networks. These data provide novel nuclear evidence to further support the notion that T. evansi and T. equiperdum should be subspecies or even strains of T. brucei.     

Zoonotic cutaneous leishmaniasis in Saudi Arabia: lesions healing naturally in man followed by a second infection with the same zymodeme of Leishmania major

A patient with a previous history of an infection with Leishmania b. braziliensis contracted zoonotic cutaneous leishmaniasis (ZCL) in the Al-Hassa oasis, Eastern Province, Saudi Arabia. Five lesions healed spontaneously over a period of 40 weeks without treatment. A year after acquiring ZCL he became infected again in the same focus. Isolates of parasites at both episodes were identified as L. major, zymodeme LON-4. Compared with the first infection of ZCL, parasites were fewer in the lesions on the second occasion, the lesions were smaller and healing was quicker (10 weeks). This work and a previous report of patients with active lesions and leishmanial scars suggest that second infections of L. major are not uncommon in the oasis where no autochthonous infections of other species of Leishmania have yet been recorded in man and only one species of Phlebotomus (P. papatasi) is known.     

Uncommon clinical presentations of cutaneous leishmaniasis in Sudan

Cutaneous leishmaniasis in Sudan is caused by Leishmania major zymodeme LON1. Self-healing usually occurs within 1 year but occasionally its duration is prolonged and treatment is required. The clinical forms are ulcers, nodules and noduloulcerative lesions. Here we describe seven patients with uncommon lesions that were difficult to recognize as Leishmania infections. These included mycetoma-like lesions, lesions that resembled L. tropica infection and others. One HIV/AIDS patient had Kaposi's sarcoma with Leishmania parasites in the Kaposi lesions. Most of these uncommon clinical forms were difficult to treat. The diagnosis depended on a high degree of suspicion and the demonstration of parasites in smears or culture. PCR was used to characterize parasites from the patients described here. Leishmania major was found by kDNA PCR in all patients, except one, who had a leishmanioma due to L. donovani. In three patients, including one with a L. tropica like-lesion, the parasites were confirmed as L. major by gp63 PCR-RFLP.     

Genomic polymorphism of Leishmania infantum: a relationship with clinical pleomorphism?

Leishmania infantum is the etiological agent of visceral (VL) and a cutaneous form (CL) of leishmaniasis around the Mediterranean Basin. In order to document the parasite genetic background corresponding to this clinical diversity, chromosome size polymorphism was analysed in 32 French isolates (18 CL and 14 VL) originating from the Cévennes and the Pyrénées Orientales (PO), and corresponding to zymodemes MON-1 and MON-29. Five chromosomes bearing tandemly repeated genes encoding for important antigens (gp63, PSA-2 and K39) or key metabolic functions (mini-exon and rDNA) were studied. Significant size variation (100-270 kbp) was observed for chromosomes bearing mini-exon, PSA-2 and rDNA genes, which involved variation in copy number of corresponding genes. The two other chromosomes showed smaller size-variation and did not involve dosage of gp63 and K39 genes. Chromosomal size showed correlation with geography and clinical origin: (i) chromosome 2 (mini-exon) was found to be significantly smaller in the PO; (ii) chromosomes 12 (PSA-2) and 27 (rDNA) were significantly smaller in the strictly cutaneous MON-29 isolates. Gene rearrangements and their synergistic effects on the phenotypic expression of the parasite are discussed.     

Leishmania donovani and cutaneous leishmaniasis, Sri Lanka

To investigate the relationship of cutaneous leishmaniasis isolates from Sri Lanka to known species, we performed DNA sequencing and microsatellite analyses. We identified Leishmania donovani as the agent of Sri Lanka cutaneous leishmaniasis and showed that these parasites are closely related to those causing visceral leishmaniasis in the Indian subcontinent.     

The American leishmaniases: some observations on their ecology and epidemiology

As the first species of Leishmania encountered were the agents of human visceral and cutaneous leishmaniasis, it is understandable that studies on these parasites for a long time concentrated on those organisms commonly causing disease in man. Epidemiological studies over the past 20 years or so, however, have led to the inescapable conclusion that the genus Leishmania is comprised of numerous species of well adapted parasites, in a wide range of mammals, throughout most of those tropical and subtropical regions of the world where phlebotomine sandflies exist (Diptera: Psychodidae). Many of the leishmanias probably never gain entrance into man: due either to an incapacity to survive in his tissues, or (more likely) because the natural sandfly vectors do not feed on him. The leishmanias that do infect man are, nevertheless, among the greatest protozoological scourges of mankind, and a better understanding of their life-cycles may well help in future prevention or control of the diseases they cause. With few exceptions the leishmaniases are zoonoses, with a major source of infection in wild or domestic animals. In the Americas, the disease is essentially a rural one, and most commonly acquired by those penetrating forested or wooded regions. The following paper deals with the better known human leishmaniases of the New World, and some new ones, and discusses the major historical events in the laborious task of elucidating their ecology and epidemiology.     

Epidemiology and diagnosis of African trypanosomiasis using DNA probes

The accurate identification of trypanosome species has been a challenging problem in the epidemiology of African trypanosomiasis, both human and animal. The last 10 years have seen great progress through the application of deoxyribonucleic acid (DNA) probe technology, although this has also revealed greater complexity than supposed. While a single repetitive DNA probe can identify all members of the subgenus Trypanosoma (Trypanozoon), including the human pathogens T. brucei gambiense and T. b. rhodesiense as well as the non-tsetse-transmitted trypanosomes T. evansi and T. equiperdum, at least 6 probes are needed to distinguish members of the subgenus Nannomonas, in which only 2 species, T. congolense and T. simiae, were previously recognized. Similarly, the subgenus Duttonella appears to consist of more than one species. Use of this battery of DNA probes to identify the trypanosomes carried by tsetse flies in the field has yielded some surprises about the accuracy (or inaccuracy) of previous identification methods. An unexpectedly high prevalence of mixed infections has been found in all the field studies carried out so far. The large number of infections that remain unidentified by the available probes suggests the existence of other, as yet unknown, trypanosome species. Limited use of the polymerase chain reaction has been made for diagnosis of human and animal trypanosomiasis, due to its high cost.     

Mixed infection with Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi in a naturally infected dog from Rio de Janeiro, Brazil

We report here the first case of co-infection with Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi in a naturally infected dog from Rio de Janeiro, Brazil. Isoenzyme characterisation identified the parasites isolated in culture from the cutaneous lesion as L. (V.) braziliensis and the isolates from blood and lymph node as L. (L.) chagasi. PCR analysis using specific primers followed by molecular hybridisation for direct Leishmania species identification in tissue fragments confirmed the presence of L. (V.) braziliensis DNA in the cutaneous lesion and of L. (L.) chagasi DNA in spleen and popliteal lymph node fragments. This report emphasises the importance of identification of Leishmania species infecting seropositive dogs in endemic areas, and the consequent re-assessment of control and epidemiological surveillance measures for the control of leishmaniasis, as is the case in Brazil.     

Synthesis, anti-Trypanosoma cruzi activity and micelle interaction studies of bisguanylhydrazones analogous to pentamidine

Three new bisguanylhydrazones analogous to pentamidine were synthesized, fully characterized and tested as anti-Trypanosoma cruzi candidates. Contrary to literature reports, that bicationic compounds are more active than monocationic compounds against Trypanosoma brucei, it was found that these bisguanylhydrazones are much less effective against T. cruzi than simple aromatic monoguanylhydrazones, thus suggesting different mechanism of action for both parasites. Spin-spin nuclear relaxation studies of the interaction of these compounds with SDS and CTAB micelles showed that only the most trypanocidal compound displays significant discrimination between anionic and cationic micelles.