RESUMO
The major surface protease (msp or gp63) of Leishmania plays a major role in the host-parasite interaction. We analysed here the structure of the msp gene locus in Leishmania (Viannia) braziliensis and compared it to results obtained in other species. Physical mapping of cosmid contigs revealed a minimum of 37 genes per haploid genome and at least 8 different msp gene families. Within the same organism, these genes showed a nucleotide sequence varying in certain stretches from 3 to 34%, and a mosaic structure. From an evolutionary point of view, major differences were observed between subgenera Viannia and Leishmania, both in terms of msp gene number and sequence. Within subgenus Viannia, phenetic analysis revealed three clusters in which sequence variants of L. (Viannia) braziliensis and L. (Viannia) guyanensis were interspersed. Functional implications of our results were explored from predicted L. (Viannia) braziliensis protein sequences: regions encoding the msp catalytic site showed a conserved sequence, while regions encoding surface domains possibly involved in the host-parasite interaction (macrophage adhesion sites and immunodominant B-cell and T-cell epitopes) were variable. We speculate that this would be an adaptive strategy of the parasite.
Assuntos
Evolução Molecular , Leishmania braziliensis/genética , Proteínas de Membrana/genética , Metaloendopeptidases/genética , Proteínas de Protozoários/genética , Animais , Variação Genética , Filogenia , Mapeamento Físico do CromossomoRESUMO
Acidic ribosomal P1 and P2b proteins, referred to as P proteins, and histone H3 are reported for first time in the Leishmania braziliensis complex. Deoxyribonucleic acid analysis and multiple sequence alignment suggest that both P proteins may maintain their structural function in the ribosomal stalk, in spite of the high rate of mutations detected. The deduced amino acid sequence of protein P1 showed 51% identity with Trypanosoma cruzi protein P1 and protein P2b showed 61% identity with T. cruzi protein P2b. Another conserved protein, L. (Viannia) braziliensis histone H3, showed 82% and 70% identity with histone H3 of L. (Leishmania) infantum and T. cruzi, respectively. The N-terminal end of this histone is divergent in comparison with the consensus eukaryotic sequence. Their predicted tridimensional structure was designed.
Assuntos
Genes de Protozoários , Leishmania braziliensis/genética , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequência Conservada , DNA de Protozoário/genética , Histonas/genética , Dados de Sequência Molecular , Fosfoproteínas/genética , Estrutura Terciária de Proteína , Proteínas Ribossômicas/genéticaRESUMO
An epidemiological study was carried out in the northern Mexican state, Nayarit. Fourteen patients with possible cutaneous leishmaniasis skin lesions gave positive Montenegro skin tests. Biopsies were taken from the skin ulcer and analyzed by polymerase chain reaction (PCR) with specific primers for the Leishmania mexicana complex; however all biopsies were not amplified. PCR carried out with specific primers for the L. braziliensis complex resulted in the amplification of all patient DNA. DNA from 12 out of 14 biopsies gave positive amplification with primers species specific for L. (Viannia) braziliensis and hybridized with a species specific L. (V.) braziliensis probe. These results demonstrate the presence in Nayarit of at least two members of the L. braziliensis complex. Most of the cutaneous lesions were caused by L. (V.) braziliensis and two by another species belonging to the L. braziliensis complex. As far as we are aware, this is the first report of L. (V.) braziliensis in Nayarit. The main risk factor associated with the contraction of this disease in Nayarit is attributed to working on coffee plantations.
Assuntos
Leishmania braziliensis , Leishmaniose Cutânea/parasitologia , Animais , Bovinos , DNA de Protozoário/isolamento & purificação , Feminino , Humanos , Leishmania braziliensis/genética , Leishmaniose Cutânea/epidemiologia , Leishmaniose Cutânea/patologia , Masculino , México/epidemiologia , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Testes CutâneosRESUMO
A new minicircle class exclusive to this specie isolated from a DNA cosmid library useful for taxonomic purposes. Experimental Parasitology 94, 143-149. In this paper we describe a new minicircle class exclusive to Leishmania (Viannia) guyanensis. The minicircle class was obtained with the aid of a total DNA cosmid library. The library was screened with an EcoRI fragment isolated from L. (V.) guyanensis (M4147). After screening seven clones were selected which showed strong hybridisation. Clones were digested and hybridised with the same probe. After hybridisation only one clone containing the desired fragment was positive. The fragment sized around 1000 bp was subcloned into pBluescript for sequencing. Sequence analysis using the GCG programme showed no substantial homology with any sequences previously reported, apart from the expected homology with the conserved region of Leishmania kDNA sequences. The probe hybridised strongly only to L. (V.) guyanensis kDNA after medium stringency washing.
Assuntos
DNA de Cinetoplasto/química , DNA de Protozoário/química , Leishmania guyanensis/genética , Animais , Sequência de Bases , Southern Blotting , Clonagem Molecular , Sequência Conservada , DNA Recombinante/química , Eletroforese em Gel de Campo Pulsado , Leishmania guyanensis/classificação , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
Two children with visceral leishmaniasis (VL), were studied by DNA analysis. DNA from liver biopsy samples from both patients, was amplified by PCR with broad primers specific for the Leishmania subgenus. DNA from the patient from Chiapas was also amplified with primers specific for the Leismania donovani complex and hybridised with a probe specific for L. donovani complex. The second patient, who is the first reported case of visceral leishmaniasis in the Mexican state of Tabasco, where localised cutaneous leishmaniasis and DCL predominate, had a co-infection with Toxoplasma gondii. The DNA from this patient was not amplified with primers specific for the L. donovani complex, did not hybridise with a probe specific for the L. donovani complex, but did hybridise with kDNA from a Mexican Leishmania mexicana strain used as a probe. We therefore, suggest that members of the L. donovani or L. mexicana complexes cause VL in Mexico.
Assuntos
Leishmania/isolamento & purificação , Leishmaniose Visceral/parasitologia , Animais , Primers do DNA , DNA de Protozoário/análise , Humanos , Lactente , Leishmania/classificação , Leishmania/genética , Leishmania donovani/genética , Leishmania infantum/genética , Leishmania mexicana/genética , Fígado/parasitologia , Masculino , México , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , Especificidade da EspécieRESUMO
A PCR-based protocol for the detection of Leishmania (Viannia) parasites in canine blood, buffy coat, and bone marrow was developed and was then tested with field samples taken from a random sample of 545 dogs from villages in Peru where Leishmania (Viannia) braziliensis and Leishmania (Viannia) peruviana are endemic. Comparative tests with cultured parasites mixed with dog blood showed that the PCR assay's sensitivity was significantly dependent on the DNA extraction protocol and the PCR primers used. Mass screening of field samples by the preferred PCR protocol detected American cutaneous leishmaniasis (ACL) in 44 of 545 (8.1%) dogs; 31 of 402 (7.7%), 20 of 223 (9.0%), and 8 of 46 (17.4%) were PCR positive when whole blood, buffy coat, and bone marrow aspirates, respectively, were tested. The high prevalence of Leishmania in both asymptomatic (7.6%) and symptomatic (18.0%) dogs provides further circumstantial evidence for their suspected role as reservoir hosts of ACL and indicates that hematogenous dissemination of parasites may be a more common pathological phenomenon than has previously been acknowledged. However, unlike for zoonotic visceral leishmaniasis, the comparatively low prevalence of Leishmania (Viannia) in the blood of symptomatic dogs indicates that PCR with blood cannot be the "gold standard" for the (mass) screening of samples in epidemiological studies.
Assuntos
Medula Óssea/parasitologia , Doenças do Cão/diagnóstico , Leishmania/isolamento & purificação , Leishmaniose Cutânea/veterinária , Parasitemia/veterinária , Animais , DNA de Protozoário/análise , DNA de Protozoário/isolamento & purificação , Doenças do Cão/parasitologia , Cães , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/epidemiologia , Leishmaniose Cutânea/parasitologia , Hibridização de Ácido Nucleico , Parasitemia/diagnóstico , Parasitemia/parasitologia , Peru , Reação em Cadeia da Polimerase/métodos , Sensibilidade e EspecificidadeRESUMO
The natural infection of sandflies by Leishmania in wild-caught specimens was studied, using the polymerase chain reaction (PCR)-hybridization technique. The PCR was carried out using 2 oligonucleotides (primers 3J1 and 3J2) derived from a repetitive nuclear DNA sequence. The primers support the enzymatic amplification of a fragment of approximately 500 bp, present in the nuclear DNA of Leishmania braziliensis. The expected band was observed in 5 of 65 sandflies containing flagellates. After hybridization with a species-specific probe, we confirmed natural infection by L. braziliensis. The technique allowed the identification of Lutzomyia gomezi and Lu. panamensis as vectors of L. braziliensis in an endemic area of cutaneous leishmaniasis in Urama, Puerto Cabello district in Venezuela. As far as we are aware, this work constitutes the first report of natural infection of Lu. panamensis with L. braziliensis in the study area. We also demonstrate that PCR-hybridization is a suitable approach to establish the Leishmania-sandfly relationship and will be useful in epidemiological studies of leishmaniasis in endemic areas.
Assuntos
Leishmania braziliensis/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Psychodidae/parasitologia , Animais , DNA de Protozoário/isolamento & purificação , Humanos , Immunoblotting/métodosRESUMO
A 1.6-kb tandem repeat sequence had previously been identified in the subtelomeric region of mini- and megabase chromosomes from Leishmania braziliensis. Southern hybridisation was used to demonstrate that the repeat is complex specific. The sequence was characterised in strains representing four species of the L. braziliensis complex. This data allowed an assessment of the evolutionary relationship of the four species. PCR primers targeted to the repeat amplify only DNA from species of the L. braziliensis complex. Titration assays indicate that a minimum of 50 fg of parasite DNA can be detected by PCR alone. Southern hybridisation increases the limit of detection to 5 fg. Interspecies variation in the repeat sequence enabled restriction enzyme digestion of PCR products to distinguish individual species within the L. braziliensis complex.
Assuntos
DNA de Protozoário/química , Leishmania braziliensis/genética , Leishmaniose Cutânea/diagnóstico , Sequências de Repetição em Tandem/genética , Telômero/genética , Animais , Sequência de Bases , Southern Blotting , Sequência Conservada , Primers do DNA/química , DNA Ribossômico/química , Leishmania braziliensis/classificação , Leishmania braziliensis/isolamento & purificação , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Alinhamento de Sequência , Especificidade da EspécieRESUMO
Leishmania parasites isolated into culture from patients with LCL or DCL from four different Mexican states were characterised using polymerase chain reaction (PCR), hybridisation with specific probes, and isoenzymes. PCR of the parasites showed that 10 of 11 of those isolates were members of the mexicana complex. This was confirmed in seven cases by isoenzymes. Restriction enzyme digests of PCR products of Mexican isolates showed the isolates to be different from the L.(L.) mexicana reference strain BEL21. Two (C2 and AM) of the isolates were shown to be a possible mixed infection between mexicana and braziliensis complex members. With a second set of samples from different patients from Campeche state, PCR of 14 biopsies indicated the presence of braziliensis complex members in six of the samples. The results showed that most of our isolates of Leishmania which come from the states of Tabasco and Veracruz are members of the Leishmania mexicana complex, but they seem to be different from the L.(L.) mexicana BEL21 reference strain. By hybridisation most of the biopsies (seven out of 14) from Campeche belong to the L. braziliensis complex and two out of 14 to L. mexicana complex and three out of 14 hybridised with both complexes, and two biopsies were negative. In Campeche, which is very close to Tabasco state and has border with Guatemala, we found members of the L. mexicana and L. braziliensis complexes.
Assuntos
DNA de Protozoário/análise , Leishmania/classificação , Leishmania/genética , Leishmaniose Cutânea/parasitologia , Reação em Cadeia da Polimerase/métodos , Animais , Southern Blotting , DNA de Cinetoplasto/análise , Humanos , Leishmania/isolamento & purificação , Leishmania braziliensis/classificação , Leishmania braziliensis/genética , Leishmania braziliensis/isolamento & purificação , Leishmania mexicana/classificação , Leishmania mexicana/genética , Leishmania mexicana/isolamento & purificação , Leishmaniose Cutânea/diagnóstico , Leishmaniose Cutânea/epidemiologia , México/epidemiologia , Sensibilidade e EspecificidadeRESUMO
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.