New sand fly (Diptera, Psychodidae) records and COI DNA barcodes in the state of Maranhão, Eastern Amazon, Brazil.

The sand fly fauna and the usefulness of the DNA barcoding fragment of the cytochrome c oxidase subunit I (COI) gene were accessed in a forest fragment in the municipality of Governador Newton Bello, state of Maranhão, Brazil. We performed entomological collections in three independent campaigns in May and October 2021, and January 2023. Sand flies were morphologically-identified and then DNA barcoded. Sequences were deposited and analyzed in the BOLD System Database, and various species delimitation algorithms, to assess whether DNA sequences merge into taxonomic units in accordance with nominal species. In total, 1,524 sand flies were collected, comprising 32 nominal species. Nyssomyia antunesi was the most abundant species (31.5 %), followed by Psychodopygus davisi (27 %). We reported for the first time in the state of Maranhão, the presence of Lutzomyia evangelistai, Lutzomyia sherlocki, Pressatia equatorialis, and Psathyromyia barrettoi. We amplified and analyzed 67 COI barcodes of 23 species, which were merged with conspecific sequences extracted from GenBank. The maximum intraspecific p distances ranged from 0.0 % to 14.74 %, while the distances to the nearest neighbor varied from 1.67 % to 13.64 %. The phylogenetic gene tree and species delimitation tools clustered sequences into well-supported clades/clusters for each nominal species, except for Pressatia choti/Pr. equatorialis, which have the lowest interspecific genetic distance (1.67 %). We sequenced for the first time COI barcodes of Brumptomyia brumpti, Evandromyia monstruosa, Micropygomyia rorotaensis, Micropygomyia pilosa, Pintomyia christenseni, Pintomyia pacae, Pr. equatorialis, Pa. barrettoi, and Psathyromyia hermanlenti, which will be useful for further molecular identification and classification proposals of Neotropical species. This study updated the current list of the sand fly fauna for the state of Maranhão to 97, and demonstrated that COI barcodes are useful for specific identification.

Trichophoromyia auraensis: evidence for cryptic species and first record in the state of Maranhão, Brazil.

Trichophoromyia auraensis (Mangabeira, 1942) (Diptera, Psychodidae, Phlebotominae) has a wide geographic distribution in the western region of the Amazon biome, where it is a putative Leishmania vector. Here, we reported for the first time a population of this species in the Brazilian state of Maranhão, in the eastern Amazon, from which we DNA-barcoded and compared with previously processed specimens from Acre State, in the western Amazon. For this, we analyzed the DNA barcoding fragment (658 bp) of the mitochondrial cytochrome c oxidase subunit I (COI) gene and the nuclear internal transcribed spacer 2 (ITS2) of Trichophoromyia species using phylogenetic gene trees, and species delimitation algorithms. The analyses of COI barcodes showed high values of genetic distance (mean K2P = 5.17) and well-supported clades/MOTUs for the eastern and western populations of T. auraensis, which may indicate a possible complex of cryptic species. The western population of this taxon merged with the close-related sand fly Trichophoromyia velezbernali Posada-López, Galvis and Galati, 2018 from Colombia, which may be associated with the recent speciation history and introgression between these populations. These evidences should be evaluated with a more comprehensive sampling in terms of analyzed populations and molecular markers.

Morphological and DNA-based description of Trichophoromyia peixotoi n. sp. (Diptera: Psychodidae), a new sand fly species from the Brazilian Amazon.

BACKGROUND: Phlebotomine sand flies of the genus Trichophoromyia Barretto, 1962 are of great relevance to public health as vectors of Leishmania protozoans. A new phlebotomine species named Trichophoromyia peixotoi n. sp. is here described based on both male morphology and COI DNA barcodes. METHODS: The sand fly specimens were collected in the Parque Nacional da Amazônia (PNA), situated in the municipality of Itaituba, state of Pará, Brazil. Morphological description was done based on 10 male specimens. Five specimens were DNA barcoded for the COI gene. RESULTS: The morphological and molecular analyses allowed the delimitation of this new species from others of Trichophoromyia. Trichophoromyia peixotoi n. sp. is closely related to other species with aedeagal ducts > 4 times the length of the sperm pump, from which it may be distinguished by the gonocoxite bristles and paramere shape. CONCLUSIONS: The description of T. peixotoi n. sp. brings the number of species of Trichophoromyia to 45, including 24 for Brazil. The integrative taxonomy effort through the analysis of COI barcodes proved to be effective in the species delimitation of some Trichophoromyia spp.

A Review of Historical Phlebotominae Taxonomy (Diptera: Psychodidae).

Psychodidae is a group constituted by ca. 3400 species distributed in six existing and one extinct subfamily. Among them, Phlebotominae is of relevant medical and veterinary interest as vectors of pathogens, such as viruses, bacteria, and trypanosomatides, to vertebrates. The taxonomy of Phlebotominae, begun in 1786, received a great boost at the beginning of the twentieth century, when some of its species began to be implicated as vectors of leishmaniasis agents. Currently, the group consists of 1060 species/subspecies described for both hemispheres. Its taxonomy and systematics have been largely based on the morphological characters of adults in view of the limited number of species known in their immature forms, as well as by molecular approaches. This review focuses on the evolution of the phlebotomine systematics, with information regarding when the descriptions of the sand fly species/subspecies occurred, where their type localities are, the number of authors involved in their descriptions, and the researchers who have most contributed to the description of these taxa and their respective institutions. The morphological characters of adult forms used in the taxonomy of groups, in an evolutionary approach, and the current state of knowledge based on immature forms is also presented.

Phlebotomine Fauna (Diptera: Psychodidae) and Infection by Leishmania spp. in Forest Fragments of a University Campus, Western Amazon.

Forest fragments consist of important ecosystems for the maintenance of sand fly populations and Leishmania hosts. This study sought to identify the phlebotomine fauna and its infection by Leishmania spp. in forest fragments on the campus of the Federal University of Acre (UFAC), Western Amazon. Monthly collections with CDC traps were carried out from March 2020 to June 2021, in four forest fragments of UFAC. Male and female insects were processed and identified at species level. A sample of females was subjected to polymerase chain reaction (PCR) analysis to verify the presence Leishmania DNA. In total, 465 specimens were collected, of which 238 were males and 227 were females. The most frequent species were Nyssomyia antunesi (Coutinho, 1939) (47.3%), Trichophoromyia sp. (Mangabeira, 1942) (18.70%), and Ny. whitmani (Antunes & Coutinho, 1939) (8.81%). Molecular analysis detected the presence of Leishmania (Ross, 1903) DNA in a specimen of Ny. antunesi, and another one of Evandromyia walker (Newstead, 1914). The forest fragments of the university campus harbor a diverse sand fly fauna with the presence of Leishmania DNA in these insects, in addition to the presence of other species considered incriminated vectors of Leishmania parasites.

Molecular taxonomy of phlebotomine sand flies (Diptera, Psychodidae) with emphasis on DNA barcoding: A review.

The taxonomy and systematics of sand flies (Diptera, Psychodidae, Phlebotominae) are one of the pillars of research aimed to identifying vector populations and the agents transmitted by these insects. Traditionally, the use of morphological traits has been the main line of evidence for the definition of species, but the use of DNA sequences is useful as an integrative approach for their delimitation. Here, we discuss the current status of the molecular taxonomy of sand flies, including their most sequenced molecular markers and the main results. Only about 37% of all sand fly species have been processed for any molecular marker and are publicly available in the NCBI GenBank or BOLD Systems databases. The genera Phlebotomus, Nyssomyia, Psathyromyia and Psychodopygus are well-sampled, accounting for more than 56% of their sequenced species. However, less than 34% of the species of Sergentomyia, Lutzomyia, Trichopygomyia and Trichophoromyia have been sampled, representing a major gap in the knowledge of these groups. The most sequenced molecular markers are those within mtDNA, especially the DNA barcoding fragment of the cytochrome c oxidase subunit I (coi) gene, which has shown promising results in detecting cryptic diversity within species. Few sequences of conserved genes have been generated, which hampers higher-level phylogenetic inferences. We argue that sand fly species should be sequenced for at least the coi DNA barcoding marker, but multiple markers with different mutation rates should be assessed, whenever possible, to generate multilocus analysis.

Preliminary observations of the urbanization and domiciliation of the American cutaneous leishmaniasis in Rio Branco, Acre, Western Amazon.

BACKGROUND: The American cutaneous leishmaniasis (ACL) is expanding in peri-urban environments. METHODS: An entomological survey was conducted in the area of the occurrence of an autochthonous urban case of ACL. Sandflies and a parasitological slide of the human case were submitted for molecular diagnosis. RESULTS: Nyssomyia whitmani and Ny. antunesi were the most frequently collected species. Ny. whitmani and Bichromomyia flaviscutellata were positive for Leishmania guyanensis and L. lainsoni, respectively. The human case tested positive for L. lainsoni. CONCLUSIONS: Sandflies and Leishmania parasites present in urban forest may occur frequently in nearby domiciliary environments; thus, these areas must be monitored.

The transmission of visceral leishmaniasis in the municipality of Guarujá, on the Coast of São Paulo state, Brazil.

OBJECTIVE: To perform an entomological survey, evaluating the circulation of Leishmania spp. in sand flies captured from the new foci of visceral leishmaniasis (VL) in the coastal region of São Paulo state. METHODS: Sand flies were captured from November 2016 to September 2018 using light traps of the Centers for Disease Control (CDC), in the neighborhood where VL cases were reported. Leishmania spp. circulation was evaluated by gut dissection and molecular analysis of the females captured. RESULTS: Nyssomyia intermedia was the more frequent species (90,7%) within the 1,203 sand flies captured. We found no flagellates in dissected females, but two pools containing females of Ny. intermedia presented DNA of L. infantum. CONCLUSION: Our results suggest that Ny. intermedia might be involved in the establishment of this new VL focus in Sao Paulo. However, before incriminating this species as a L. infantum vector, further studies should investigate other vectorial capacity parameters, including competence, survival, and feeding habits.

Xenodiagnosis in four domestic cats naturally infected by Leishmania infantum.

Leishmaniasis is a neglected tropical disease that continues to pose a serious public health problem. Albeit dogs have long been held as the major reservoirs of Leishmania infantum, the involvement of domestic cats in the zoonotic cycle of visceral leishmaniasis has gained prominence. Here, 240 cats were evaluated by clinical signs and haematological/biochemical changes compatible with leishmaniasis and were diagnosed by serological, molecular, and parasitological techniques. Thus, four cats naturally infected by L. infantum were submitted to xenodiagnosis. A total of 203 females of Lutzomyia longipalpis were subjected to feeding on four cats, with all females completing the blood meal. Parasitological and molecular assays were carried out to evaluate the presence of L. infantum in the sand flies' midgut. Promastigotes were observed in 10 females (6.5%) that fed on one cat, and L. infantum DNA was detected in 17 (8.4%) females that fed on two cats. Our results strengthen the evidence that naturally infected cats are capable of transmitting L. infantum to sand flies.

Preliminary observations of the urbanization and domiciliation of the American cutaneous leishmaniasis in Rio Branco, Acre, Western Amazon

ABSTRACT Background: The American cutaneous leishmaniasis (ACL) is expanding in peri-urban environments. Methods: An entomological survey was conducted in the area of the occurrence of an autochthonous urban case of ACL. Sandflies and a parasitological slide of the human case were submitted for molecular diagnosis. Results: Nyssomyia whitmani and Ny. antunesi were the most frequently collected species. Ny. whitmani and Bichromomyia flaviscutellata were positive for Leishmania guyanensis and L. lainsoni, respectively. The human case tested positive for L. lainsoni. Conclusions: Sandflies and Leishmania parasites present in urban forest may occur frequently in nearby domiciliary environments; thus, these areas must be monitored.

The transmission of visceral leishmaniasis in the municipality of Guarujá, on the Coast of São Paulo state, Brazil

ABSTRACT OBJECTIVE To perform an entomological survey, evaluating the circulation of Leishmania spp. in sand flies captured from the new foci of visceral leishmaniasis (VL) in the coastal region of São Paulo state. METHODS Sand flies were captured from November 2016 to September 2018 using light traps of the Centers for Disease Control (CDC), in the neighborhood where VL cases were reported. Leishmania spp. circulation was evaluated by gut dissection and molecular analysis of the females captured. RESULTS Nyssomyia intermedia was the more frequent species (90,7%) within the 1,203 sand flies captured. We found no flagellates in dissected females, but two pools containing females of Ny. intermedia presented DNA of L. infantum. CONCLUSION Our results suggest that Ny. intermedia might be involved in the establishment of this new VL focus in Sao Paulo. However, before incriminating this species as a L. infantum vector, further studies should investigate other vectorial capacity parameters, including competence, survival, and feeding habits.

Remarkable diversity, new records and Leishmania detection in the sand fly fauna of an area of high endemicity for cutaneous leishmaniasis in Acre state, Brazilian Amazonian Forest.

The species richness of Amazonian phlebotomines is considered to be one of the highest in the world. In the present study, we investigated the richness and diversity of phlebotomine fauna in Xapuri city, Acre state, Western Brazilian Amazonia, which is an area that is highly endemic for cutaneous leishmaniasis. Sand fly collections were performed monthly from August 2013 to July 2015 (288 h total of sampling effort) in intradomiciliary, peridomiciliary, and forested environments of two localities. Collected females were dissected, microscopically examined for flagellates in their guts, and preserved in ethanol. A total of 21,197 specimens comprising 14 genera and 57 species were collected, and the majority of these were Nyssomyia, Psychodopygus, and Trichophoromyia genera. Three new records of phlebotomine species for Acre are presented here, including Brumptomyia brumpti, Psathyromyia pradobarrientosi, and for the first time in Brazil, Th. omagua. In Xapuri, the phlebotomine fauna of different ecotopes was varied in regard to abundance, diversity, and frequency, and they included proven and permissive vectors of Leishmania spp. The fauna discovered in the forested areas (57 species) was richer and more diverse than was that (33 species) identified in the peri­ and intra-domiciles. The identification of Leishmania subgenera that were present in sand fly guts according to SSU rRNA sequences revealed ten and three species harboring Leishmania of subgenera Viannia and Leishmania (most likely Leishmania amazonensis), respectively. The presence of Leishmania (Leishmania) in sand flies are reported here for the first time in Acre. The presence of L. (Viannia) spp. in Brumptomyia sp. and Lutzomyia sherlocki. and the occurrence of mixed infections with Leishmania of both subgenera in Ps. lainsoni have been reported for the first time in Brazil. Taken together, data from previous studies and from the present study highlight the remarkable complexity of phlebotomine fauna that is possibly due to the well-preserved Xapuri forested areas sustaining vital economic activities of plant extraction and ecological tourism. Our findings also provide new insights into the ongoing adaptation of Trichophoromyia and Psychodopygus species to human habitats.

Natural infection of Lutzomyia longipalpis (Cembrene-1 population) with Leishmania infantum in a new visceral leishmaniasis focus in the eastern region of São Paulo State, Brazil.

INTRODUCTION: Visceral leishmaniasis (VL) transmission has been associated with two different populations of the Lutzomyia longipalpis complex in São Paulo state. METHODS: In a recent focus of VL, we captured and dissected sand flies and investigated Leishmania infantum infection by parasitological, PCR, and sequencing analysis. RESULTS: Flagellates were observed in 2 of 47 (4.2%) cembrene-1 Lu. longipalpis females. The sequences obtained matched those of Le. infantum. CONCLUSIONS: We found that the transmission of Le. infantum by cembrene-1 females may occur at a high rate in this focus of VL and presented new data on the vector capacity of this population.

Detection of Pintomyia fischeri (Diptera: Psychodidae) With Leishmania infantum (Trypanosomatida: Trypanosomatidae) Promastigotes in a Focus of Visceral Leishmaniasis in Brazil.

Visceral leishmaniasis is spreading in Brazil where the main vector of its agent, Leishmania infantum Nicolle, 1908, is the Lutzomyia longipalpis (Lutz & Neiva, 1912) species complex (Diptera: Psychodidae: Phlebotominae), on which many of the activities of the visceral leishmaniasis surveillance program are based. However, there are areas where canine, and/or human cases have been occurring without the presence of this species complex as in the western part of the Greater São Paulo Metropolitan region, where Embu das Artes municipality is situated. In this area, Pintomyia fischeri (Pinto, 1926) has been implicated as potential vector of Le. infantum but so far its natural infection with this parasite has not yet been ascertained. Therefore, the present study sought to investigate the natural infection in sand flies of a CVL focus in Embu das Artes. The sand fly collections were undertaken with Shannon and CDC traps, monthly, between 1800 and 2100 hours from November 2018 to October 2019, inclusive. A total of 951 sand flies (457 males and 494 females), belonging to 10 species, were captured. Pintomyia fischeri was the predominant species (89.5%); of which 426 females were dissected and one of them (0.23%) was found to be harboring flagellates in its midgut. A sample of these flagellates was isolated in culture and characterized by a 234 base pair fragment of Leishmania heat-shock protein 70 gene (hsp70) and restriction fragment length polymorphism with Hae III restriction enzyme as Le. infantum. This finding reinforces previous evidence of Pi. fischeri as a vector of Le. infantum in foci of visceral leishmaniasis and highlights the importance of vector surveillance in areas where this species occurs.

Natural infection of Lutzomyia longipalpis (Cembrene-1 population) with Leishmania infantum in a new visceral leishmaniasis focus in the eastern region of São Paulo State, Brazil

Abstract INTRODUCTION Visceral leishmaniasis (VL) transmission has been associated with two different populations of the Lutzomyia longipalpis complex in São Paulo state. METHODS In a recent focus of VL, we captured and dissected sand flies and investigated Leishmania infantum infection by parasitological, PCR, and sequencing analysis. RESULTS Flagellates were observed in 2 of 47 (4.2%) cembrene-1 Lu. longipalpis females. The sequences obtained matched those of Le. infantum. CONCLUSIONS We found that the transmission of Le. infantum by cembrene-1 females may occur at a high rate in this focus of VL and presented new data on the vector capacity of this population.

First report of an Onchocercidae worm infecting Psychodopygus carrerai carrerai sandfly, a putative vector of Leishmania braziliensis in the Amazon.

Sandflies are insects of public health interest due to their role as vectors of parasites of the genus Leishmania, as well as other pathogens. Psychodopygus carrerai carrerai is considered an important sylvatic vector of Leishmania (Viannia) braziliensis in Amazonia. In this study, sandflies were collected in a forested area in the Xapuri municipality, in the State of Acre (Northern Brazil). Two Ps. carrerai carrerai females were found parasitized with a larval form of a filarial worm, one in the labium of the proboscis, the other after the head was squashed, suggesting they were infective larvae. Sandflies were identified through morphological characters as well as amplification and sequencing of the cytochrome oxidase gene (COI). This was the first sequence obtained for Ps. carrerai carrerai for this marker. The obtained nematodes were also characterized through direct sequencing of a fragment of COI and 12S genes, both mitochondrial, and ITS1, a nuclear marker. Phylogenetic analyses revealed that the filarial nematodes belong to a species without sequences for these markers in the database, part of family Onchocercidade and closely related to genus Onchocerca (12S tree). Although sandfly infection with nematodes including members of the Onchocercidae has been reported in the Old World, this is the first report of sandfly infection by a member of the Onchocercidae family in the New World, to the best of our knowledge. Considering that the phylogenetic relationships and location in the insect, it can be expected that this is a parasite of mammals and the transmission cycle should be clarified.

Phlebotominae (Diptera: Psychodidae) and Biomes in the State of Mato Grosso do Sul, Brazil.

The state of Mato Grosso do Sul, Brazil is an important region of South America regarding leishmaniasis, with the great diversity of sandflies reported since 1938 and wide biodiversity represented by its biomes (Atlantic Forest, Cerrado, and Pantanal). A checklist of sandflies is presented here for the state and respective biomes. The data base was compiled from primary and secondary data. The primary data collection involved automatic light traps at Miranda-Abobral Pantanal subregion and in subregion Baixa Nhecolância of Pantanal. The secondary data were obtained from entomology scientific collections and a literature review of articles from 1938 to 2019, including entomological museum collections. A total of 71 species were reported, belonging to 14 genera and 13 subgenera, in 61 municipalities of the state, including the type locality of 9 species, and the first report of Lutzomyia cruzi (Mangabeira 1938), vector of Leishmania infantum (Nicolle 1937), in the Pantanal region of Miranda-Abobral. Other vector species, Bichromomyia flaviscutellata (Mangabeira 1942), Lutzomyia longipalpis (Lutz & Neiva 1912), Nyssomyia antunesi (Coutinho 1939), Nyssomyia intermedia (Lutz & Neiva 1912), Nyssomyia neivai (Pinto 1926), and Nyssomyia whitmani (Antunes & Coutinho 1939), are reported in the entire area of the state. Maps containing the distribution of sandflies on Mato Grosso do Sul biomes were produced. The eclecticism of the species in relation to their ecotypes was observed, with several species using the three biome types as habitats and breeding sites.

Scanning Electron Microscopy of Sand Flies of the Chagasi Series, Psychodopygus (Diptera: Psychodidae) Genus, Focusing on the Genitalia.

Males of cryptic or closely related species present great morphological variation in their genitalia, whereas females, such as those of the Chagasi Series of the Psychodopygus Mangabeira, 1941 genus, are more similar. Therefore, our aim was to study the fine structure of the male genitalia of five species of the Chagasi Series to better understand the variation in their morphology and its influence on the copulatory process. The sand fly species were captured in the following Brazilian states: Psychodopygus chagasi (Costa Lima, 1941) (Rondônia), Psychodopygus complexus (Mangabeira, 1941) (Tocantins), Psychodopygus squamiventris maripaensis (Floch & Abonnenc, 1946) (Amapá), Psychodopygus squamiventris squamiventris (Lutz & Neiva, 1912) (Amazonas), and Psychodopygus wellcomei Fraiha, Shaw & Lainson, 1971 (Pará and Ceará). Insects were stored in ethanol 70% (then dehydrated) and dry after they were sputtered with gold. The samples were observed under a scanning electron microscope. Microtrichiae, two types of trichoid sensilla, coeloconic and chaetic sensillae, were observed on the antenna of all species, with no difference between them. Only on the anepimeron of P. squamiventris squamiventris a modified 'racket'-like scale was observed. As for the male genitalia, the setae and structures of each species were fully described, such as the small setae on the paramere apex of the P. squamiventris subspecies, and the grooves present in this region and on the paramere lobe of P. complexus and P. wellcomei, which are impossible to observe with optic microscopy. New information is thus provided on the male genitalia, which can contribute to future bionomic studies of these species.

Phlebotomine (Diptera: Psychodidae) fauna in a cavern containing cave paintings and its surrounding environment, Central-West Brazil.

The interior of caves usually presents climate, light, soil, flora and fauna that distinguish it from the external environment. Among the sandfly species found in caves, several may act as vectors of Leishmania spp. and others are a nuisance to humans due to their painful bites. This study sought to identify the phlebotomine fauna in a cavern with cave paintings ("Gruta do Pitoco") which attract tourists in Alcinopólis municipality, Mato Grosso do Sul state, Brazil. The sandflies were collected from March 2014 to September 2015, with automatic light traps, installed monthly, in the entrance and interior of the cave and outside it. A total of 7645 specimens belonging to 27 species were collected. Lutzomyia dispar, Martinsmyia oliverai and those of the Lu. longipalpis complex (Lu. cruzi and Lu. longipalpis) were the predominant species. Among the three environments sampled, no great difference between species richness was observed. Comparing these environments, the interior of the cave presented the lowest diversity index due to the high frequency of Lu. dispar (46%). For the first time, specimens (only females) of the genus Deanemyia were collected in Mato Grosso do Sul state. Moreover, the study revealed the sympatric occurrence of Lu. cruzi and Lu. longipalpis, both proven vectors of L. infantum, and also Lu. dispar, a highly anthropophilic species, which often causes annoyance to those who visit the cave.

Leishmania (Viannia) braziliensis type 2 as probable etiological agent of canine cutaneous leishmaniasis in Brazilian Amazon.

Canine cutaneous leishmaniasis (CCL) is a zoonosis of public health interest, and in the Americas, Leishmania (Viannia) braziliensis has been identified as the main etiological agent. The present study sought to investigate Leishmania spp. infection in domestic dogs from a rural area of the Xapuri municipality, Acre state, Brazilian Amazonia. For this purpose, visits were carried out to domiciles where the human cases of American cutaneous leishmaniasis (ACL) occurred, followed by the clinical evaluation of the animals in search of clinical signs suggestive of CCL. Blood samples were collected from 40 dogs, 13 of which had lesions suggestive of CCL, and biopsies of these lesions were performed. The methods used were Neal, Novy, and Nicolle's (NNN) medium cultures and direct parasitological examination. Further, to detect and characterize Leishmania DNA some molecular techniques were performed such as conventional polymerase chain reaction (PCR) and sequencing targeting SSU rDNA and ITS1, restriction fragment length polymorphism (RFLP) and high resolution melting (HRM) analysis targeting hsp70. The investigation revealed that the results obtained from the parasitological methods were negative. In PCR by ITS1 and network topology sequences, six strains from dogs, isolated from the Peruvian Andes, appeared identical to Leishmania (Viannia) braziliensis type 2 (99-100%). By other molecular methods these samples turned out to be positive to Leishmania (Viannia) sp.. The diagnosis of Leishmania in domestic dogs from Acre state showed a high proportion of infected animals, and the occurrence of L. braziliensis type 2 in Brazil for the first time. This new report suggests that L. braziliensis type 2 is both trans- and cis-Andean. However, more studies are needed regarding the clinical and diagnostic aspects of this species of Leishmania.