Numerical zymotaxonomy and variability of the internal transcribed spacers (ITS) between the small and large subunits of the rRNA genes were used to examine strain variation and relationships in natural populations of Leishmania (Viannia) braziliensis. A total of 101 strains from distinct hosts and Brazilian geographic regions were assigned to 15 zymodemes clustered in two major genetic groups. The great number of isolates (48.5%) placed in zymodeme IOC/Z-27 were collected on the Atlantic coast. The high molecular diversity found in populations in the Amazon Basin was related to the great number of sandfly vector(s) in that region. The results of the restriction fragment length polymorphism analysis of the ITS depicted considerable intraspecific variation. Genotypic groups A, B, and C contained 39, 40, and 22 isolates, which were divided into 16, 10, and 15 genotypes, respectively. The genetic polymorphism observed demonstrates the degree of diversity of L. Infections with the parasitic protozoan Leishmania (Viannia) braziliensis Vianna 1911 (Kinetoplastida: Trypanosomatidae) or strain variants are recognized as causing human illness in many areas of (sub)tropical America (at least 15 countries), where it constitutes a significant public health problem. Many of these parasites seem to have a unique life cycle, with different phlebotomine sandfly (Diptera: Psychodidae) vectors and/or animal reservoirs and a different geographic distribution (13). This pathogen is capable of producing a variety of clinical manifestations, such as (i) cutaneous leishmaniasis (CL), which may heal spontaneously; (ii) mucosal leishmaniasis (ML), a hyperergic invasive ulcerative form that progresses in the absence of any apparent cellular defect (15); and (iii) disseminated CL (4).Most of the environmental factors affecting the epidemiology of the various leishmaniases are still poorly understood. Wild mammals serve as reservoirs for most of the New World Leishmania spp. (21), but there is increasing evidence that some of the human pathogenic Leishmania strains can be maintained in both sylvan and urban cycles. In the case of L. (V.) braziliensis, the principal vertebrate hosts in the sylvan cycle have not been identified, but there is evidence that dogs, horses, and donkeys may serve as reservoir hosts of this parasite (15). The existence of an urban cycle involving peridomestic sandfly species for L. (V.) braziliensis reflects the ability of these parasites and their vectors to adapt to changes in their original forested habitats with important public health implications. Studies using molecular techniques to characterize L. (V.) braziliensis populations from different regions have shown a relationship between level of similarity among the parasite populations (12, 24) and their geographic range, but recent data have also indicated that the considerable variability detected among these parasites is more probably related to the sandfly vector(s) and/or animal reservoir(s) involved in the transmission cycles (18).Pathogens that produce...

Evidence of Leishmania infection was found in small mammals captured between 1996 and 2000 in the Amaraji region, Pernambuco State, Brazil. The kDNA polymerase chain reaction (PCR), using primers specific for subgenus L. (Viannia), was positive for 43/153 water rats (Nectomys squamipes), 13/81 black rats (Rattus rattus), 15/103 grass mice (Bolomys lasiurus), 1/14 marsh mice (Holochilus scieurus), 2/50 field mice (Akodon arviculoides), 2/12 woolly opossums (Marmosa sp.), and 5/37 common opossums (Didelphis albiventris). This same kDNA PCR was positive for 12/61 dog and 8/58 horse skin samples. In paired PCR tests of 203 small mammals, 18.7% were positive with the kDNA primers and 18.2% with rDNA primers. Amastigotes were seen in 26/460 and L. (V.) braziliensis was isolated from 5 grass mice and 1 black rat. We concluded that small mammals, particularly rodents, are infected with parasites of the subgenus L. (Viannia). The isolation of L. (V.) braziliensis zymodeme IOC/Z74 from 6 rodents and the fact that all the other described L. (Viannia) species that commonly infect humans have never been found in rodents or marsupials leads us to suggest that the positive PCRs indicate infections of L. (V.) braziliensis. The isolation of zymodeme IOC/Z74 from humans reinforces our hypothesis that small, ground-loving mammals, such as rodents are the primary reservoirs of L. (V.) braziliensis.

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This study reinforces the involvement of different small mammals (e.g., N. squamipes, R. rattus and H. scieurus) in the transmission cycles of L. (V.) braziliensis and L. (L.) infantum in north-eastern Brazil. The finding of L. (V.) braziliensis infection in black rats suggests a rapid process of adaptation of a New World Leishmania species to an Old World rodent and raises interesting questions regarding the co-evolution of these parasites and their vertebrate hosts.

Summaryobjectives To identify the aetiological agents of cutaneous leishmaniasis and to investigate the genetic polymorphism of Leishmania (Viannia) parasites circulating in an area with endemic cutaneous leishmaniasis (CL) in the Atlantic rainforest region of northeastern Brazil.methods Leishmania spp. isolates came from three sources: (i) patients diagnosed clinically and parasitologically with CL based on primary lesions, secondary lesions, clinical recidiva, mucocutaneous leishmaniasis and scars; (ii) sentinel hamsters, sylvatic or synanthropic small rodents; and (iii) the sand fly species Lutzomyia whitmani. Isolates were characterised using monoclonal antibodies, multilocus enzyme electrophoresis (MLEE) and polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region rDNA locus.results Seventy-seven isolates were obtained and characterised. All isolates were identified as Leishmania (Viannia) braziliensis serodeme 1 based on reactivity to monoclonal antibodies. MLEE identified 10 zymodemes circulating in the study region. Most isolates were classified as zymodemes closely related to L. (V.) braziliensis, but five isolates were classified as Leishmania (Viannia) shawi. All but three of the identified zymodemes have so far been observed only in the study region. Enzootic transmission and multiclonal infection were observed.conclusions Our results confirm that transmission cycle complexity and the co-existence of two or more species in the same area can affect the level of genetic polymorphism in a natural Leishmania population. Although it is not possible to make inferences as to the modes of genetic exchange, one can speculate that some of the zymodemes specific to the region are hybrids of L. (V.) braziliensis and L. (V.) shawi.keywords Leishmania, leishmaniasis, MLEE, molecular typing, L. (V.) braziliensis, L. (V.) shawi

PCR-based approaches targeting kinetoplast DNA were evaluated for the diagnosis of American cutaneous leishmaniasis (ACL) in regions of endemicity in northeastern Brazil. A total of 119 cutaneous biopsy specimens from patients with ACL and nonleishmaniasis cutaneous lesions were studied. Two PCR-based systems were used; one was specific for the subgenus Viannia, and the other was specific for the genus Leishmania. The PCR specific for the subgenus Viannia had a sensitivity of 95.4%, whereas the genus-specific PCR detected the target DNA in 88.2% of the samples tested. The specificities of the assays, determined with samples from a group with nonleishmaniasis cutaneous lesions, was 100%. The results of the conventional tests indicate that the sensitivities of the PCR-based methods were significantly higher than those of smear examination, histological staining, and isolation by culture (P < 0.05). Antibodies specific for Leishmania braziliensis were detected by indirect immunofluorescence in 82.9% of the patients tested. Parasites were isolated from 40 of 86 patients (46.5%). Sixty-seven percent of dermal scrapings and 66.2% of stained tissue sections were positive by microscopy. Amplified products from the subgenus-specific PCR hybridized with the Leishmania panamensis minicircle, confirming infection consistent with L. braziliensis. The evidence available at present incriminates L. braziliensis as the only causative agent of ACL in the state of Pernambuco in Brazil.

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