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In the present study, we tested 391 fleas collected from guinea pigs (Cavia porcellus) (241 Pulex species, 110 Ctenocephalides felis, and 40 Tiamastus cavicola) and 194 fleas collected from human bedding and clothing (142 Pulex species, 43 C. felis, five T. cavicola, and four Ctenocephalides canis) for the presence of Bartonella DNA. We also tested 83 blood spots collected on Flinders Technology Associates (FTA) cards from guinea pigs inhabiting 338 Peruvian households. Bartonella DNA was detected in 81 (20.7%) of 391 guinea pig fleas, in five (2.6%) of 194 human fleas, and in 16 (19.3%) of 83 guinea pig blood spots. Among identified Bartonella species, B. rochalimae was the most prevalent in fleas (89.5%) and the only species found in the blood spots from guinea pigs. Other Bartonella species detected in fleas included B. henselae (3.5%), B. clarridgeiae (2.3%), and an undescribed Bartonella species (4.7%). Our results demonstrated a high prevalence of zoonotic B. rochalimae in households in rural areas where the research was conducted and suggested a potential role of guinea pigs as a reservoir of this bacterium.

Approximately 20 Leishmania species are known to cause cutaneous, mucocutaneous, and visceral disorders in humans. Identification of the causative species in infected individuals is important for appropriate treatment and a favorable prognosis because infecting species are known to be the major determinant of clinical manifestations and may affect treatments for leishmaniasis. Although Leishmania species have been conventionally identified by multilocus enzyme electrophoresis, genetic analysis targeting kinetoplast and nuclear DNA (kDNA and nDNA, respectively) is now widely used for this purpose. Recently, we conducted countrywide epidemiological studies of leishmaniasis in Ecuador and Peru to reveal prevalent species using PCR-RFLP targeting nDNA, and identified unknown hybrid parasites in these countries together with species reported previously. Furthermore, comparative analyses of kDNA and nDNA revealed the distribution of parasites with mismatches between these genes, representing the first report of mito-nuclear discordance in protozoa. The prevalence of an unexpectedly high rate (~10%) of genetically complex strains including hybrid strains, in conjunction with the observation of mito-nuclear discordance, suggests that genetic exchange may occur more frequently than previously thought in natural Leishmania populations. Hybrid Leishmania strains resulting from genetic exchanges are suggested to cause more severe clinical symptoms when compared with parental strains, and to have increased transmissibility by vectors of the parental parasite species. Therefore, it is important to clarify how such genetic exchange influences disease progression and transmissibility by sand flies in nature. In addition, our aim was to identify where and how the genetic exchange resulting in the formation of hybrid and mito-nuclear discordance occurs.

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mannose phosphate isomerase (mpi) gene was applied to 134 skin samples collected from patients with cutaneous leishmaniasis (CL) in Peru for identification of the infecting parasite at the species level, and the results were compared with those of cytochrome b (cyt b) gene sequencing obtained in previous studies. Although most results (121/134) including 4 hybrids of Leishmania (Viannia) braziliensis and L. (V.) peruviana corresponded to those obtained in the previous study, PCR-RFLP analyses revealed the distribution of putative hybrid strains between L. (V.) peruviana and L. (V.) lainsoni in two samples, which has never been reported. Moreover, parasite strains showing discordance between kinetoplast and nuclear genes (kDNA and nDNA), so-called mito-nuclear discordance, were identified in 11 samples. Of these, six strains had the kDNAs of L. (V.) braziliensis or L. (V.) peruviana and nDNAs of L. (V.) guyanensis, and three strains had the kDNAs of L. (V.) shawi and nDNAs of L. (V.) braziliensis. The rest were identified as mito-nuclear discordance strains having kDNAs of L. (V.) braziliensis or L. (V.) peruviana and nDNAs of L. (V.) lainsoni, and kDNAs of L. (V.) lainsoni and nDNAs of L. (V.) braziliensis. The results demonstrate that Leishmania strains in Peru are genetically more complex than previously considered., Author summary Protozoan parasites of the genus Leishmania are able to undergo genetic exchange during their growth. The previous description of hybrids in Peru and the recent discovery of unexpected genetically complex strains having characteristics of both hybrid and mito-nuclear discordance in its neighbouring country (Ecuador) with a similar eco-epidemiological situation led us to consider that the genetic structure of Leishmania strains in Peru is more complicated than previously thought. In an effort to revise the data on Leishmania strain dispersion in Peru and to search for evidence of genetic recombination, the present study was conducted. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis targeting the mannose phosphate isomerase (mpi) gene sequence was performed to identify the infecting parasite at the species level in 134 skin samples collected from patients with cutaneous leishmaniasis (CL) in Peru, and the results were compared with those of cytochrome b (cyt b) gene sequencing obtained in previous studies. Most results (121/134) including 4 hybrids between L. (V.) braziliensis and L. (V.) peruviana showed agreement between PCR-RFLP of the mpi gene and cyt b gene sequence analysis; however, 13 of 134 samples revealed the distribution of strains with hybrids and mito-nuclear discordance. The results demonstrate that genetically complex Leishmania strains are present in Peru. These findings indicate that Leishmania strain dispersion in Peru is genetically more complex than previously considered. Further prospective studies including larger samples and the isolation of parasite strains are required to update the available data.

This study provides comprehensive information on the past and current status of the Andean cutaneous leishmaniasis (Andean-CL, uta) in Peru and Ecuador, mainly focusing on the causative Leishmania parasites and clinico-epidemiological features. Available information and data including our unpublished works were analyzed thoroughly. Endemic regions of the Andean-CL (uta) in Peru run from the north Piura/Cajamarca to the south Ayacucho at a wide range of the Pacific watersheds of the Andes through several departments, while in Ecuador those exist at limited and spotted areas in the country's mid-southwestern two provinces, Azuay and Chimborazo. The principal species of the genus Leishmania are completely different at subgenus level, L. (Viannia) peruviana in Peru, and L. (Leishmania) mexicana and L. (L.) major-like (infrequent occurrence) in Ecuador. The Peruvian uta is now prevalent in different age and sex groups, being not clearly defined as found in the past. The precise reasons are not known and should be elucidated further, though probable factors, such as emergence of other Leishmania parasites, non-immune peoples' migration into the areas, etc., were discussed briefly in the text. The Andean-CL cases in Ecuador are more rural than before, probably because of a rapid development of the Leishmania-positive communities and towns, and the change of life-styles of the inhabitants, including newly constructed houses and roads in the endemic areas. Such information is helpful for future management of the disease, not only for Leishmania-endemic areas in the Andes but also for other endemic areas.

To obtain further insight into geographic distribution of Leishmania species in Peru, a countrywide survey, including central to southern rainforest areas where information on causative parasite species is limited, was performed based on cytochrome b (cyt b) and mannose phosphate isomerase (mpi) gene analyses. A total of 262 clinical samples were collected from patients suspected of cutaneous leishmaniasis (CL) in 28 provinces of 13 departments, of which 99 samples were impregnated on FTA (Flinders Technology Associates) cards and 163 samples were Giemsa-stained smears. Leishmania species were successfully identified in 83 (83.8%) of FTA-spotted samples and 59 (36.2%) of Giemsa-stained smear samples. Among the 142 samples identified, the most dominant species was Leishmania (Viannia) braziliensis (47.2%), followed by L. (V.) peruviana (26.1%), and others were L. (V.) guyanensis, L. (V.) lainsoni, L. (V.) shawi, a hybrid of L. (V.) braziliensis and L. (V.) peruviana, and Leishmania (Leishmania) amazonensis. Besides the present epidemiological observations, the current study provided the following findings: 1) A hybrid of L. (V.) braziliensis and L. (V.) peruviana is present outside the Department of Huanuco, the only place reported, 2) Many cases of CL due to L. (V.) lainsoni, an uncommon causative species in Peru, were observed, and 3) L. (V.) shawi is widely circulating in southern Amazonian areas in Peru., Author summary Leishmaniasis, a neglected tropical disease (NTD) caused by the intracellular protozoa of the genus Leishmania, affects at least 12 million people in 96 countries. Peru is one of the most highly endemic countries for cutaneous leishmaniasis (CL), and our previous study identified Leishmania (Viannia) braziliensis, L. (V.) peruviana, and L. (V.) guyanensis in the tropical rainforest, in the Andean highlands, and in the northern and central rainforest areas, respectively, as the main CL-causative agents. In addition, distribution of L. (V.) lainsoni, L. (V.) shawi, a hybrid of L. (V.) braziliensis and L. (V.) peruviana, and Leishmania (Leishmania) amazonensis has been identified. Of these, one case each of L. (V.) shawi infection was reported from the Departments of Junin and Madre de Dios, while clinical cases due to the hybrid of L. (V.) braziliensis and L. (V.) peruviana were recorded only in the Department of Huanuco. To further elucidate the current geographic distribution of causative Leishmania species in Peru, a countrywide survey, including central to southern rainforest areas where little information on causative parasites is available, was performed based on the cytochrome b (cyt b) gene sequence and PCR-RFLP analysis of the mannose phosphate isomerase (mpi) gene by using FTA (Flinders Technology Associates) card-spotted samples and smear slides as DNA sources. In addition to current epidemiological observations, the current study revealed that 1) A hybrid of L. (V.) braziliensis and L. (V.) peruviana was identified, for the first time, outside the Department of Huanuco, 2) L. (V.) lainsoni, an uncommon CL-causative species in Peru, was predominantly found in the Department of Puno, where causative Leishmania species are not well-studied, and 3) New endemic foci of L. (V.) shawi were identified in central to southern rainforest areas of Peru.

The natural infection of sand flies by Leishmania was investigated in Andean areas located between the Central and Eastern Cordilleras of northern Peru where cutaneous leishmaniasis caused by Leishmania (Viannia) peruviana is endemic. Sand flies were captured at five locations along the Utcubamba River in the Department of Amazonas, and morphologically identified under a microscope. Among 422 female sand flies dissected, the most dominant species was Pintomyia verrucarum (320 flies), followed by Pi. maranonensis (83 flies), Pi. robusta (13 flies), and Lutzomyia castanea (6 flies). Genetic analysis of sand flies from these areas together with those from other areas revealed that individuals of Pi. verrucarum were closely related regardless of morphological variation of their spermathecae. On the other hand, individuals of Pi. maranonensis collected in the study area were distant from those of other areas with genetic distances over the intraspecific level but mostly below the interspecific level, suggesting the unique characteristics of sand flies in this area. The natural infection of sand flies by flagellate parasites was detected mainly in the hindgut of each one of Pi. verrucarum and Pi. maranonensis. Both parasite species were identified as L. (V.) peruviana based on cytochrome b and mannose phosphate isomerase gene analyses. In addition, parasite species obtained from the lesion of a patient with cutaneous leishmaniasis in the study area in this period was identified as L. (V.) peruviana. These results strongly suggest that Pi. verrucarum and Pi. maranonensis are responsible for the transmission of L. (V.) peruviana in these areas. This is the first report of the natural infection of Pi. maranonensis by L. (V.) peruviana., Author summary Phlebotomine sand flies are tiny insects of the family Psychodidae in the order Diptera, and female sand flies suck blood for egg production. Approximately 1,020 sand fly species have been recorded in the world, of which about 550 species are in the New World. Only a part of them are associated with medically important infectious diseases such as leishmaniasis, and importantly, each vector species transmits specific species of Leishmania. Since the infecting Leishmania species is the major determinant of the clinical outcome and its endemicity is largely dependent on the prevalence of the vector species, the identification of circulating sand flies and vector species, which determine transmissible parasite species, is important to predict the risk and expansion of the disease in endemic and surrounding areas. However, the vector species involved in disease transmission remains unidentified in most endemic areas because the infection rate in sand fly populations is very low. In the present study, sand flies were investigated in the Department of Amazonas in the Eastern Andes of northern Peru, in which cutaneous leishmaniasis caused by Leishmania (Viannia) peruviana is endemic, to clarify the transmission mechanism of leishmaniasis in these areas. In addition, genetic analyses of circulating sand flies were performed to elucidate the characteristics of sand flies in these areas.

Leishmaniasis remains one of the world's most neglected diseases, and early detection of the infectious agent, especially in developing countries, will require a simple and rapid test. In this study, we established a quick, one-step, single-tube, highly sensitive loop-mediated isothermal amplification (LAMP) assay for rapid detection of Leishmania DNA from tissue materials spotted on an FTA card. An FTA-LAMP with pre-added malachite green was performed at 64 degrees C for 60 mm using a heating block and/or water bath and DNA amplification was detected immediately after incubation. The LAMP assay had high detection sensitivity down to a level of 0.01 parasites per mu l. The field- and clinic-applicability of the colorimetric FTA-LAMP assay was demonstrated with 122 clinical samples collected from patients suspected of having cutaneous leishmaniasis in Peru, from which 71 positives were detected. The LAMP assay in combination with an FTA card described here is rapid and sensitive, as well as simple to perform, and has great potential usefulness for diagnosis and surveillance of leishmaniasis in endemic areas. (C) 2015 Elsevier B.V. All rights reserved.

Background; The goal of this study was to reassess the taxonomic status of A. maculatum, A. triste and A. tigrinum by phylogenetic analysis of five molecular markers [four mitochondrial: 12S rDNA, 16S rDNA, the control region (DL) and cytochrome c oxidase 1 (cox1), and one nuclear: ribosomal intergenic transcribed spacer 2 (ITS2)]. In addition, the phenotypic diversity of adult ticks identified as A. maculatum and A. triste from geographically distinct populations was thoroughly re-examined. Results: Microscopic examination identified four putative morphotypes distinguishable by disjunct geographical ranges, but very scant fixed characters. Analysis of the separated mitochondrial datasets mostly resulted in conflicting tree topologies. Nuclear gene sequences were almost identical throughout the geographical ranges of the two species, suggesting a very recent, almost explosive radiation of the terminal operational taxonomic units. Analysis of concatenated molecular datasets was more informative and indicated that, although genetically very close to the A. maculatum - A. triste lineage, A. tigrinum was a monophyletic separate entity. Within the A. maculatum - A. triste cluster, three main clades were supported. The two morphotypes, corresponding to the western North American and eastern North American populations, consistently grouped in a single monophyletic clade with many shared mitochondrial sequences among ticks of the two areas. Ticks from the two remaining morphotypes, south-eastern South America and Peruvian, corresponded to two distinct clades. Conclusions: Given the paucity of morphological characters, the minimal genetic distance separating morphotypes, and more importantly the fact that two morphotypes are genetically indistinguishable, our data suggest that A. maculatum and A. triste should be synonymized and that morphological differences merely reflect very recent local adaptation to distinct environments in taxa that might be undergoing the first steps of speciation but have yet to complete lineage sorting. Nonetheless, future investigations using more sensitive nuclear markers and/or crossbreeding experiments might reveal the occurrence of very rapid speciation events in this group of taxa. Tentative node dating revealed that the A. tigrinum and A. maculatum - A. triste clades split about 2 Mya, while the A. maculatum - A.triste cluster radiated no earlier than 700,000 years ago. EEA Rafaela Fil: Lado, Paula. Georgia Southern University. Institute for Coastal Plain Science. United States National Tick Collection; Estados Unidos Fil: Nava, Santiago. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina Fil: Mendoza-Uribe, Leonardo. Instituto Nacional de Salud. Laboratorio de Entomología; Perú Fil: Cáceres, Abraham G. Universidad Nacional Mayor de San Marcos. Facultad de Medicina. Departamento Académico de Microbiología Médica; Perú. Instituto Nacional de Salud. Laboratorio de Entomología; Peru Fil: Delgado de la Mora, Jesus. University of Sonora. Department of Medicine and Health Sciences; México Fil: Licona Enriquez, Jesus D. University of Sonora. Department of Medicine and Health Sciences; México Fil: Delgado de la Mora, David. Technologic Institute of Sonora. Department of Agronomic and Veterinary Sciences; México Fil: Labruna, Marcelo B. Universidade de São Paulo. Faculdade de Medicina Veterinária e Zootecnia. Departamento de Medicina Veterinária Preventiva e Saúde Animal; Brasil Fil: Durden, Lance A. Georgia Southern University. Department of Biology; Estados Unidos Fil: Allerdice, Michelle E. J. Centers for Disease Control and Prevention. National Center for Emerging and Zoonotic Infectious Diseases. Rickettsial Zoonoses Branch; Estados Unidos Fil: Paddock, Christopher D. Centers for Disease Control and Prevention. National Center for Emerging and Zoonotic Infectious Diseases. Rickettsial Zoonoses Branch; Estados Unidos Fil: Szabó, Matías P.J. Universidade Federal de Uberlandia. Faculdade de Medicina Veterinaria e Zootecnia. Laboratório de Ixodologia; Brasil Fil: Venzal, José Manuel. Universidad de la República. CENUR Litoral Norte-Salto. Facultad de Veterinaria. Departamento de Parasitología Veterinaria; Uruguay Fil: Guglielmone, Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; Argentina Fil: Beati, Lorenza. Georgia Southern University. Institute for Coastal Plain Science. United States National Tick Collection; Estados Unidos

In the present study, we investigated 238 fleas collected from cats and dogs in three regions of Peru (Ancash, Cajamarca, and Lima) for the presence of Bartonella DNA. Bartonella spp. were detected by amplification of the citrate synthase gene (16.4%) and the 16S-23S intergenic spacer region (20.6%). Bartonella rochalimae was the most common species detected followed by Bartonella clarridgeiae and Bartonella henselae. Our results demonstrate that dogs and cats in Peru are infested with fleas harboring zoonotic Bartonella spp. and these infected fleas could pose a disease risk for humans.

An analysis of reported cases of cutaneous leishmaniasis (CL) was performed using the data registered in the southern Ecuadorian Amazon region during 27 years from 1986 to 2012. The cases/subjects with both the suspected CL lesions and the amastigote-positive results were recruited for the analysis. The yearly occurrence of cases showed a markedly higher number during the six years, 1988 and 1993. After 1994 when the insecticide spraying campaign using helicopter in 1993-1994, the number dropped remarkably. Then, the yearly occurrence gradually fluctuated from 101 cases in 1996 to 11 in 2009, maintaining a low number of cases after the campaign. The monthly occurrence of cases showed a markedly high number during March and August, suggesting a correlation to the rainy season (months) in the areas. A statistical significance was found between the monthly average number of the CL case and the average precipitation (p=0.01474). It was suggested that the time of transmission of CL would depend on the rainy seasons at each endemic area of Ecuador, which has a diverse climatic feature depending on the geographic regions. Such information at given leishmaniasis-endemic areas of Ecuador would be important for the future planning of the disease control. Molecular analysis and characterization of clinical samples revealed the presence of Leishmania (Viannia) braziliensis.

Phlebotomine sand flies are the only proven vectors of leishmaniases, a group of human and animal diseases. Accurate knowledge of sand fly species identification is essential in understanding the epidemiology of leishmaniasis and vector control in endemic areas. Classical identification of sand fly species based on morphological characteristics often remains difficult and requires taxonomic expertise. Here, we generated DNA barcodes of the cytochrome c oxidase subunit 1 (COI) gene using 159 adult specimens morphologically identified to be 19 species of sand flies, belonging to 6 subgenera/species groups circulating in Peru, including the vector species. Neighbor-joining (NJ) analysis based on Kimura 2-Parameter genetic distances formed non-overlapping clusters for all species. The levels of intraspecific genetic divergence ranged from 0 to 5.96%, whereas interspecific genetic divergence among different species ranged from 8.39 to 19.08%. The generated COI barcodes could discriminate between all the sand fly taxa. Besides its success in separating known species, we found that DNA barcoding is useful in revealing population differentiation and cryptic diversity, and thus promises to be a valuable tool for epidemiological studies of leishmaniasis. Copyright (C) 2015 Published by Elsevier B.V. All rights reserved.

Haplotype and gene network analyses were performed on mitochondrial cytochrome oxidase I and cytochrome b gene sequences of Lutzomyia (Lu.) ayacuchensis populations from Andean areas of Ecuador and southern Peru where the sand fly species transmit Leishmania (Leishmania) mexicana and Leishmania (Viannia) peruviana, respectively, and populations from the northern Peruvian Andes, for which transmission of Leishmania by Lu. ayacuchensis has not been reported. The haplotype analyses showed higher intrapopulation genetic divergence in northern Peruvian Andes populations and less divergence in the southern Peru and Ecuador populations, suggesting that a population bottleneck occurred in the latter populations, but not in former ones. Importantly, both haplotype and phylogenetic analyses showed that populations from Ecuador consisted of clearly distinct clusters from southern Peru, and the two populations were separated from those of northern Peru. Crown Copyright (C) 2014 Published by Elsevier B.V. All rights reserved.

The vector Lutzomyia sand flies and reservoir host mammals of the Leishmania parasites, causing the Andean cutaneous leishmaniasis (Andean-CL, uta) in Peru and Ecuador were thoroughly reviewed, performing a survey of literatures including our unpublished data. The Peruvian L. (V.) peruviana, a principal Leishmania species causing Andean-CL in Peru, possessed three Lutzomyia species, Lu. peruensis, Lu. verrucarum and Lu. ayacuchensis as vectors, while the Ecuadorian L. (L.) mexicana parasite possessed only one species Lu. ayacuchensis as the vector. Among these, the Ecuadorian showed a markedly higher rate of natural Leishmania infections. However, the monthly and diurnal biting activities were mostly similar among these vector species was in both countries, and the higher rates of infection (transmission) reported, corresponded to sand fly's higher monthly-activity season (rainy season). The Lu. tejadai sand fly participated as a vector of a hybrid parasite of L. (V.) braziliensis/L. (V.) peruviana in the Peruvian Andes. Dogs were considered to be principal reservoir hosts of the L. (V.) peruviana and L. (L.) mexicana parasites in both countries, followed by other sylvatic mammals such as Phyllotis andium, Didelphis albiventris and Akodon sp. in Peru, and Rattus rattus in Ecuador, but information on the reservoir hosts/mammals was extremely poor in both countries. Thus, the Peruvian disease form demonstrated more complicated transmission dynamics than the Ecuadorian. A brief review was also given to the control of vector and reservoirs in the Andes areas. Such information is crucial for future development of the control strategies of the disease.

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.

A PCR-Restriction Fragment Length Polymorphism (RFLP) targeting the mannose phosphate isomerase gene was established to differentiate Leishmania species distributed near the Department of Huanuco, Peru. The technique was applied to 267 DNA samples extracted from Giemsa-stained smears of cutaneous lesions taken from patients suspected for cutaneous leishmaniasis in the area, and the present status of causative Leishmania species was identified. Of 114 PCR-amplified samples, 22, 19, 24 and 49 samples were identified to be infected by Leishmania (Viannia) braziliensis, L. (V.) peruviana, L. (V.) guyanensis, and a hybrid of L. (V.) braziliensis/L. (V.) peruviana, respectively, and the validity of PCR-RFLP was confirmed by sequence analysis. Since PCR-RFLP is simple and rapid, the technique will be a useful tool for the epidemiological study of leishmaniasis.

A total of 103 ticks, collected from canines, horses, donkeys, and snakes from Peru, were screened for the presence of Bartonella DNA by polymerase chain reaction analysis. Bartonella DNA was detected in two ticks using Bartonella 16S-23S intergenic spacer region primers and in an additional two ticks using Bartonella NADH dehydrogenase gamma subunit gene (nuoG) primers. Bartonella rochalimae Eremeeva et al., B. quintana Schmincke, and B. elizabethae Daly et al. DNA was detected in a Rhipicephalus sanguineus Latreille (Acari: Ixodidae) female tick removed from a dog and B. quintana DNA was present in a Dermacentor nitens Neumann (Acari: Ixodidae) pool of five larvae, one nymph, and one adult male tick collected from donkeys. This is the first study to report the detection of B. rochalimae, B. quintana, and B. elizabethae DNA in ticks from Peru. Further investigations must be performed to decipher the role ticks may play in the transmission of Bartonella species.

Within the sand fly genus Lutzomyia, the Verrucarum species group contains several of the principal vectors of American cutaneous leishmaniasis and human bartonellosis in the Andean region of South America. The group encompasses 40 species for which the taxonomic status, phylogenetic relationships, and role of each species in disease transmission remain unresolved. Mitochondrial cytochrome c oxidase I (COI) phylogenetic analysis of a 667-bp fragment supported the morphological classification of the Verrucarum group into series. Genetic sequences from seven species were grouped in well-supported monophyletic lineages. Four species, however, clustered in two paraphyletic lineages that indicate conspecificity--the Lutzomyia longiflocosa-Lutzomyia sauroida pair and the Lutzomyia quasitownsendi-Lutzomyia torvida pair. COI sequences were also evaluated as a taxonomic tool based on interspecific genetic variability within the Verrucarum group and the intraspecific variability of one of its members, Lutzomyia verrucarum, across its known distribution.

The natural infection of sand flies by Leishmania species was studied in the Andean areas of Peru where cutaneous leishmaniasis caused by Leishmania (Viannia) peruviana is endemic. Sand flies were captured by human bait and Center for Disease Control (CDC) light trap catches at Nambuque and Padregual, Department of La Libertad, Peru, and morphologically identified. Among 377 female sand flies dissected, the two dominant man-biting species were Lutzomyia (Helcocyrtomyia) peruensis (211 flies) and Lutzomyia (Helcocyrtomyia) caballeroi (151 flies). Another sand fly species captured by light trap was Warileya phlebotomanica (15 flies). The natural infection of sand flies by flagellates was detected in 1.4% of Lu. (H.) peruensis and 2.6% of Lu. (H.) caballeroi, and the parasite species were identified as Le. (V.) peruviana and Trypanosoma avium, respectively, by molecular biological methods. The results indicated that the vector species responsible for the transmission of leishmaniasis in the study areas is Lu. (H.) peruensis. In addition, the presence of Trypanosoma in man-biting sand fly species means that more careful consideration is necessary for vector research in areas of Andean Peru where leishmaniasis is endemic.

In this study, each of 60 rRNA internal transcribed spacer (ITS) 1 and ITS2 sequences was determined from 44 individuals of 14 morphologically identified New World sand fly Lutzomyia species in Ecuador, and their interspecies and intraspecies genetic diversity was compared. Distinguishing between related species based on the ITS1 sequence was difficult because of variability, while the genetic diversity of ITS2 was distinct even among closely related species. Further, an assessment of intraspecies ITS sequence diversity in the subgenus Helcocyrtomyia revealed no correlation between sequence variation and geographic distribution. The results strongly suggested ITS2 to be a more suitable marker than ITS1 for the taxonomic analysis of Lutzomyia species including closely related species. Moreover, neither ITS sequence may be useful for the analysis of population structures in Lutzomyia species.

The natural infection of sand flies by Leishmania was examined in the Department of Huanuco of Peru, where cutaneous leishmaniasis caused by a hybrid of Leishmania (Viannia) braziliensis/L. (V.) peruviana is endemic. A total of 2,997 female sand flies were captured by CDC light traps and Shannon traps, of which 2,931 and 66 flies were identified as Lutzomyia tejadai and Lu fischeri, respectively. Using crude DNA extracted from individual sand flies as a template, Leishmania DNA was detected from one Lu. tejadai. The parasite species was identified as a hybrid of L. (V.) braziliensis/L. (V.) peruviana on the basis of cytochrome b and mannose phosphate isomerase gene analyses. The result suggested that Lu. tejadai is responsible for the transmission of the hybrid Leishmania circulating in this area., Author Summary Leishmaniasis is a protozoan disease caused by members of the genus Leishmania, which are distributed worldwide, especially in tropical and subtropical areas. More than 20 species of Leishmania are described as causative agents of human leishmaniases and clinical features are largely associated with the infective species. The parasites are transmitted by bites of female sand flies of the genus Phlebotomus in the Old World and Lutzomyia in the New World. Approximately 800 sand fly species have been recorded; of these, fewer than 10% have been confirmed as vectors of Leishmania and only a restricted number of species transmit specific Leishmania species. Therefore, sand fly surveillance is important for predicting risk and spread of leishmaniasis in endemic and surrounding areas. Cutaneous leishmaniasis caused by a hybrid of L. (Viannia) braziliensis/L. (V.) peruviana have been reported in the Department of Huanuco of Peru; however, the vector species has not been identified. In the present study, approximately 3,000 sand flies were examined, and the sand fly infected by the hybrid Leishmania strain was successfully identified.

A reassessment of the taxonomic status of Amblyomma cajennense based on the morphological analyses of ticks from the whole distribution area of the species resulted in the redescription of A. cajennense, the validation of 2 species which had been reduced to synonymy in the past, Amblyomma mixtum and Amblyomma sculptum, and the description and definition of 3 new species, Amblyomma tonelliae n. sp., Amblyomma interandinum n. sp., and Amblyomma patinoi n. sp. This study provides descriptions and redescriptions, scanning electron microscopic and stereomicroscopic images, updated synonymies, information on geographical distributions, and host associations for each of the 6 species. Amblyomma cajennense s.s. is found in the Amazonian region of South America, A. interandinum is reported from the northern part of the Inter-Andean valley of Peru, A. mixtum is present from Texas (U.S.A.) to western Ecuador, A. patinoi occurs in the Eastern Cordillera of Colombia, A. tonelliae is associated with the dry areas of the Chaco region which spans from central-northern Argentina to Bolivia and Paraguay, whereas A. sculptum is distributed from the humid areas of northern Argentina, to the contiguous regions of Bolivia and Paraguay and the coastal and central-western states of Brazil. Fil: Nava, Santiago. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Lorenza, Beati. Georgia Southern University. Institute for Coastal Plain Science; Estados Unidos Fil: Labruna, Marcelo B.. Universidade de Sao Paulo; Brasil Fil: Cáceres, Abraham G.. Universidad Nacional Mayor de San Marcos; Perú Fil: Mangold, Atilio Jose. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Guglielmone, Alberto Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; Argentina

En localidades ubicadas entre los distritos de San José de Lourdes (05°03'Longitud Sur), provinda de San Ignacio y Lonya Grande (06°05'30" Longitud Sur), provinda de Utcubamba, Region Nor Orietital del Maranón, donde la bartonellosis humana o Verruga Peruana es endémica, se capturaron 2774 flebótomos entre 1987 y 1992; identificándo se 13 especies; 11 correspondeu al género Lutzomyia, 1 a Brumptomyia y 1 a Warileya. De las especies capturadas, Zutzomyia sp. similar a Lutzomyia serrana y Lutzomyia maranonensis sp. n., Galati, Cáceres & Le Pont, son las mas abundantes (89.74%), por lo que, se especula que puedeit ser consideradas vectores potenciales de la Verruga Peruana. Se reporta Lutzomyia verrucarum (Townsend, 1913) por primem vez para la provinda de Utcubamba.Two thousand seven hundred and seventy four sand flies were captured since 1987 to 1992 in endemic areas of human bartonellosis and Peruvian Verruga (district of San José de Lourdes, province of San Ignacio - 0903' LS, and district of Lonya Grande, province of Utcubamba - 06°05'30" LS) belonging to the Northeast region of Maranon, Peru. Thirteen species were identified, 11 belonging to the genus Lutzomyia, 1 to Brumptomyia and the other to Warileya. Lutzomyia sp, close to L. serrana and L. maranonensis n. sp., Galati, Cáceres & Le Pont are more prevalent (89.74% of the individuals captured). Suggests working of the kind. Lutzomyia verrucarum (Townsend, 1913) is reported in Utcubamba prownce for the first time.

The genetic divergence caused by genetic drift and/or selection is suggested to affect the vectorial capacity and insecticide susceptibility of sand flies, as well as other arthropods. In the present study, cytochrome b (cyt b) gene sequences were determined in 13 species circulating in Peru to establish a basis for analysis of the genetic structure, and the intraspecific genetic diversity was assessed in the Lutzomyia (Lu.) peruensis, a main vector species of Leishmania (Viannia) peruviana in Peruvian Andes. Analysis of intraspecific genetic diversity in the cyt b gene sequences from 36 Lu. peruensis identified 3 highly polymorphic sites in the middle region of the gene. Haplotype and gene network analyses were performed on the cyt b gene sequences of 130 Lu. peruensis in 9 Andean areas from 3 Departments (Ancash, Lima and La Libertad). The results showed that the populations of La Libertad were highly polymorphic and that their haplotypes were distinct from those of Ancash and Lima, where dominant haplotypes were observed, suggesting that a population bottleneck may have occurred in Ancash and Lima, but not in La Libertad. The present study indicated that the middle region of the cyt b gene is useful for the analysis of genetic structure in sand fly populations.

The population genetic structure of Lutzomyia verrucarum (Townsend), a sand fly disease vector of Carrion's disease and cutaneous leishmaniasis in the Peruvian Andes, was characterized by sequencing 653 bp of cytochrome b and 1,125 bp of the NADH dehydrogenase subunit 4 genes of its mitochondrial genome. DNA sequence variation within and between valleys was compared in a sample of 220 sand flies from three valleys (Purisima, Huaylas, and Conchucos) and five departments (Amazonas, Cajamarca, Piura, Lima, and Huancavelica). Gene network and phylogenetic analyses indicated a high similarity of haplotypes collected within a single valley (0–0.52% nucleotide divergence). Flies from each valley had unique genotypes not shared with specimens from other valleys or from more distant regions (0.8–3.1% nucleotide divergence). Mountain ranges and geographic distance appear to have impeded migration (Nm=

The female of the phlebotomine sand fly Lutzomyia reclusa Fernandez & Rogers 1991 [= Pintomyia (Pifanomyia) reclusa (Fernandez & Rogers) sensu Galati], is described for the first time, based on specimens collected in the Department of Cajamarca, in northern Peru. The female can be recognized from other species of the series pia, species group Verrucarum, by wing venation with beta shorter than half of alpha, labrum just shorter than head width but longer than flagellomere 1, palpomere 5 much longer than palpomere 3, arrangement of cibarial armature, and form of spermathecae and relative size of spermathecal ducts. Diagnostic characters and measurements of the male of Lu. reclusa are provided as well.

Lutzomyia verrucarum (Townsend, 1913) (Diptera: Psychodidae), vector natural de la verruga peruana o enfermedad de Carrión es una especie propia del Perú. Su distribución geográfica esta entre los paralelos 5º y 13º25' de latitud Sur, se encuentra en los valles Occidentales e Interandinos de los Andes. La distribución altitudinal de Lu. verrucarum en los diversos valles es variable; asi: Occidentales, desde 1100 hasta 2980 msnm e Interandinos, de 1200 a 3200 msnm. En ciertas áreas verrucógenas no hay correlación entre la presencia de Lu. verrucarum y la enfermedad de Carrión lo que suguiere la existencia de vectores secundarios. Lutzomyia verrucarum (Townsend, 1913) (Diptera: Psychodidae); the natural vector of Bartonella bacilliformis, agent of human bartonellosis (peruvian verruga or Carrion's disease), is a native specie of Peru; its geographic distribution occurres between latitudes 5º and 13º 25' South: in the Occidental and Interandean valleys of the Andean. The altitudinal distribution of Lu. verrucarum in the different valleys is as follows: Occidental between 1100 and 2980 m sea level and Interandean from 1200 to 3200 m sea level. Some discrepancies between the distribution of Carrion's disease and Lu. verrucarum suggest the existence of secondary vectors in certain areas where Lu. verrucarum is not present

Forty-one Leishmania peruviana isolates were selected along a north-south transect which crossed areas endemic for uta in three different biogeographical regions in the Peruvian Andes. The isolates were analysed by molecular karyotyping and hybridization with three chromosome-derived DNA probes. All the isolates could be distinguished from L. braziliensis by their pLb-134 hybridization patterns. However, the patterns with the other probes (pLb-168 and -22) could be used to cluster the Peruvian isolates in discrete groups (karyodemes) which varied in their level of similarity with L. braziliensis. The geographical distribution of these karyodemes supports the hypothesis that eco-graphical isolation has contributed to the heterogeneity of L. peruviana.

The study objective was to investigate an acute case of Chagas disease in the San Pedro de Shishita community, Pebas District, in the Peruvian Amazon basin, a non-endemic area. Both parents of the index case (acute case) were thoroughly interviewed, a seroepidemiological survey was carried out in the community, parasitological exams were carried out only in relatives of the index case, and triatomine bugs were searched for inside houses, peridomiciliary, and in wild environments. Seroprevalence for IgG anti-T. cruzi antibodies was 1/104 (0.96%), using an ELISA test and an indirect immunofluorescence assay. Panstrongylus geniculatus and Rhodnius pictipes adults were found. The index case is autochthonous from San Pedro de Shishita, but the source of transmission is unknown. O objetivo deste trabalho foi estudar caso da doença de Chagas aguda na comunidade indígena de San Pedro de Shishita, sem conhecimento da origem da transmissão. San Pedro de Shishita, distrito Pebas, região da Amazônia peruana é uma área não endêmica. Foram entrevistados os pais do paciente e feito inquérito soroepidemiológico dos participantes e estudos parasitológicos da família e procurou-se também triatomíneos no ambiente doméstico, peridomiciliar e silvestre. A soroprevalência de anticorpos IgG anti-T. cruzi foi 1/ 104 (0,96%) por ELISA e imunofluorescência indireta. Foram encontrados adultos de Panstrongylus geniculatus e Rhodnius pictipes

Sand flies from the Andean areas of Ecuador and Peru were examined for Leishmania infections by using our recently established molecular mass screening method. Leishmanial minicircle DNA-positive sand flies were detected in 3 of 192 and 1 of 462 samples from Ecuador and Peru, respectively. Sand fly species were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the 18S ribosomal RNA (rRNA) gene, and the positive flies were Lutzomyia (Lu.) ayacuchensis and Lu. peruensis, respectively. Furthermore, cytochrome b and mannose-phosphate isomerase gene sequence analyses identified the parasites from Ecuador and Peru as Leishmania (Leishmania) mexicana and L. (Viannia) peruviana, respectively. Thus, the mass screening method was confirmed to be a powerful tool for sand fly research.

The southernmost limit of the distribution of endemic Andean cutaneous leishmaniasis (CL), commonly known as Uta, is localized in the western Andean valleys of Ayacucho, Peru. This area is completely isolated from other regions endemic for this disease. Identification of the insect vector for Andean CL was carried out by combining entomologic and parasitologic approaches. Two Lutzomyia species were captured: Lutzomyia ayacuchensis and Lu. noguchii. The former species was considered responsible for transmission of Leishmania because 1) there was a coincidence in space and time between the presence of this insect and the distribution of Andean CL, 2) it was shown to be highly anthropophilic, 3) Leishmania parasites of the subgenus Viannia were detected by a specific polymerase chain reaction assay, 4) promastigotes isolated from this insect were shown by multilocus enzyme electrophoresis and molecular karyotyping to belong to the same deme of Leishmania (Viannia) peruviana as the one circulating in humans living in the study area, and 5) the complete cycle of L. (V.) peruviana was observed in experimental infections of Lu. ayacuchensis. Parasite and vector homogeneity found in Ayacucho contrasted with the heterogeneity reported for other areas endemic for Andean CL. The potential influence of ecologic determinants on this geographically isolated area is discussed.

Lutzomyia spp. are New World phlebotomine sand flies, many of which are involved in the transmission of human diseases, such as leishmaniases and bartonellosis. The systematic classification of the approximately 400 species in the genus has been based on morphological characters, but the relationships within the genus are still very much in question. We have inferred phylogenies of 32 species of phlebotomine sand flies belonging to seven sub-genera and two species groups, by using fragments of the mitochondrial small subunit (12SrRNA) and of the nuclear large subunit (28SrRNA) ribosomal gene sequences. The subgenus Helcocyrtomyia and the Verrucarum species group, prominent representatives of the Peruvian sand fly fauna, were represented by 11 and 7 species, respectively. Although based on a limited number of taxa, the resulting phylogenies, based on 837 characters, provide an initial phylogenetic backbone for the progressive reconstruction of infrageneric relationships within Lutzomyia.

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 sampled 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.

One of the objectives of a clinical and epidemiological study of Andean leishmaniasis (uta) in the Huayllacallán valley, Ancash, Perú, was to identify the vector(s) of its causative agent, Leishmania (Viannia) peruviana Vélez, 1913...