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1.
Rev Bras Parasitol Vet ; 29(4): e017820, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33295378

RESUMO

Leishmania infantum infection in cats has been reported in several countries, including Brazil. However, the transmission of visceral leishmaniasis (VL) from cats to another host has not been proven yet. Therefore, the aim of this study was to verify the possibility of L. infantum transmission from cats to dogs. In order to verify the possibility of VL transmission from the cat to the dog, xenodiagnosis was carried out in a VL-positive cat, using 55 female Lutzomyia longipalpis. Five days later, 40 insects were dissected to verify Leishmania infection. The remaining 15 females were fed in a healthy dog. The potential infection of the dog was verified through clinical, serological, parasitological examinations, and PCR, at three, six, and twelve months post-infection. All 55 L. longipalpis females became visibly engorged. Leishmania promastigotes were detected in 27.5% of the dissected insects. Leishmania infection in the dog was confirmed upon first evaluation. DNA sequencing of the parasite isolated from the cat confirmed L. infantum infection and showed 99% similarity with the L. infantum DNA sequences from the dogs. Through this study, it was possible to confirm the L. infantum experimental transmission from a domestic cat to a domestic dog through its biological vector L. longipalpis.


Assuntos
Doenças do Gato , Doenças do Cão , Leishmania infantum , Leishmaniose Visceral , Animais , Brasil , Doenças do Gato/parasitologia , Doenças do Gato/transmissão , Gatos , DNA de Protozoário/química , Doenças do Cão/parasitologia , Doenças do Cão/transmissão , Cães , Feminino , Insetos Vetores/parasitologia , Leishmania infantum/genética , Leishmaniose Visceral/transmissão , Leishmaniose Visceral/veterinária , Psychodidae/parasitologia
2.
Mem Inst Oswaldo Cruz ; 115: e200157, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33206821

RESUMO

BACKGROUND: In Acre state, Brazil, the dissemination of cutaneous leishmaniasis has increased in recent years, with limited knowledge of the potential Leishmania spp. vectors involved. OBJECTIVES: Here, data concerning the sandfly fauna of Brasiléia municipality, Leishmania DNA-detection rates and the identification of blood meal sources of insects captured in 2013-2015 are presented. METHODS: Parasite detection in female sandflies was performed individually by multiplex polymerase chain reaction (PCR) (Leishmania kDNA/sandfly cacophony-gene), with the identification of Leishmania spp. by hsp70-PCR and sequencing. The identification of blood gut-content from fed females was performed by cyt b-PCR and sequencing. FINDINGS: A total of 4,473 sandflies were captured. A subgroup of 864 non-blood-fed females evaluated for the presence of Leishmania DNA showed 2.9% positivity for Leishmania (Viannia) braziliensis and L. (V.) guyanensis. The identification of blood meal sources was performed in 96 blood-fed females, allowing the identification of 13 vertebrate species. In nine/96 fed females, DNA from L. (V.) shawi, L. (V.) guyanensis, L. (V.) braziliensis and Endotrypanum sp. was detected. MAIN CONCLUSIONS: In Brumptomyia sp. and Evandromyia termitophila, the first report of Leishmania DNA-detection is provided in Acre; Nyssomyia shawi is implicated as potential vector of L. (V.) braziliensis and L. (V.) guyanensis for the first time in Brazil.


Assuntos
DNA/análise , Insetos Vetores/genética , Leishmania/genética , Psychodidae/parasitologia , Animais , Brasil , DNA de Protozoário/análise , Feminino , Insetos Vetores/classificação , Insetos Vetores/parasitologia , Leishmania/isolamento & purificação , Leishmaniose Cutânea/transmissão , Reação em Cadeia da Polimerase , Psychodidae/classificação
3.
Sci Rep ; 10(1): 17664, 2020 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-33077743

RESUMO

Leishmaniasis encompasses a spectrum of diseases caused by a protozoan belonging to the genus Leishmania. The parasite is transmitted by the bite of sand flies, which inoculate the promastigote forms into the host's skin while acquiring a blood meal. Nyssomyia neivai is one of the main vectors of tegumentary leishmaniasis (TL) in Brazil. Southeastern Brazil is an endemic region for TL but also overlaps with an endemic focus for pemphigus foliaceus (PF), also known as Fogo Selvagem. Salivary proteins of sand flies, specifically maxadilan and LJM11, have been related to pemphigus etiopathogenesis in the New World, being proposed as an environmental trigger for autoimmunity. We present a comprehensive description of the salivary transcriptome of the N. neivai, using deep sequencing achieved by the Illumina protocol. In addition, we highlight the abundances of several N. neivai salivary proteins and use phylogenetic analysis to compare with Old- and New-World sand fly salivary proteins. The collection of protein sequences associated with the salivary glands of N. neivai can be useful for monitoring vector control strategies as biomarkers of N. neivai, as well as driving vector-vaccine design for leishmaniasis. Additionally, this catalog will serve as reference to screen for possible antigenic peptide candidates triggering anti-Desmoglein-1 autoantibodies.


Assuntos
Insetos Vetores/genética , Psychodidae/genética , Proteínas e Peptídeos Salivares/genética , Animais , Brasil , Feminino , Perfilação da Expressão Gênica , Biblioteca Gênica , Insetos Vetores/parasitologia , Leishmaniose Cutânea/transmissão , Leishmaniose Mucocutânea/transmissão , Masculino , Pênfigo/etiologia , Filogenia , Psychodidae/parasitologia , Glândulas Salivares/metabolismo , Análise de Sequência de RNA
4.
PLoS Negl Trop Dis ; 14(10): e0008666, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33052941

RESUMO

The microbial consortium associated with sandflies has gained relevance, with its composition shifting throughout distinct developmental stages, being strongly influenced by the surroundings and food sources. The bacterial components of the microbiota can interfere with Leishmania development inside the sandfly vector. Microbiota diversity and host-microbiota-pathogen interactions regarding New World sandfly species have yet to be thoroughly studied, particularly in Lutzomyia longipalpis, the primary vector of visceral leishmaniasis in Brazil.The native microbiota of different developmental stages and physiological conditions of Lu. longipalpis (Lapinha Cave), was described by culturing and 16s rRNA gene sequencing. The 16s rRNA sequencing of culture-dependent revealed 13 distinct bacterial genera (Bacillus, Enterococcus, Erwinia, Enterobacter, Escherichia, Klebsiella, Lysinibacillus, Pseudocitrobacter, Providencia, Pseudomonas, Serratia, Staphylococcus and Solibacillus). The in vitro and in vivo effects of each one of the 13 native bacteria from the Lu. longipalpis were analyzed by co-cultivation with promastigotes of L.i. chagasi, L. major, L. amazonensis, and L. braziliensis. After 24 h of co-cultivation, a growth reduction observed in all parasite species. When the parasites were co-cultivated with Lysinibacillus, all parasites of L. infantum chagasi and L. amazonensis died within 24 hours. In the in vivo co-infection of L.chagasi, L. major and L. amazonensis with the genera Lysinibacillus, Pseudocitrobacter and Serratia it was possible to observe a significant difference between the groups co-infected with the bacterial genera and the control group.These findings suggest that symbiont bacteria (Lysinibacillus, Serratia, and Pseudocitrobacter) are potential candidates for paratransgenic or biological control. Further studies are needed to identify the nature of the effector molecules involved in reducing the vector competence for Leishmania.


Assuntos
Bactérias/classificação , Leishmania/fisiologia , Psychodidae/microbiologia , Psychodidae/parasitologia , Animais , Microbiota , RNA Bacteriano/genética , RNA Ribossômico 16S/genética
5.
PLoS Pathog ; 16(10): e1008494, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33091070

RESUMO

The shape and form of the flagellated eukaryotic parasite Leishmania is sculpted to its ecological niches and needs to be transmitted to each generation with great fidelity. The shape of the Leishmania cell is defined by the sub-pellicular microtubule array and the positioning of the nucleus, kinetoplast and the flagellum within this array. The flagellum emerges from the anterior end of the cell body through an invagination of the cell body membrane called the flagellar pocket. Within the flagellar pocket the flagellum is laterally attached to the side of the flagellar pocket by a cytoskeletal structure called the flagellum attachment zone (FAZ). During the cell cycle single copy organelles duplicate with a new flagellum assembling alongside the old flagellum. These are then segregated between the two daughter cells by cytokinesis, which initiates at the anterior cell tip. Here, we have investigated the role of the FAZ in the morphogenesis of the anterior cell tip. We have deleted the FAZ filament protein, FAZ2 and investigated its function using light and electron microscopy and infection studies. The loss of FAZ2 caused a disruption to the membrane organisation at the anterior cell tip, resulting in cells that were connected to each other by a membranous bridge structure between their flagella. Moreover, the FAZ2 null mutant was unable to develop and proliferate in sand flies and had a reduced parasite burden in mice. Our study provides a deeper understanding of membrane-cytoskeletal interactions that define the shape and form of an individual cell and the remodelling of that form during cell division.


Assuntos
Citoesqueleto/metabolismo , Flagelos/fisiologia , Interações Hospedeiro-Parasita , Leishmania/crescimento & desenvolvimento , Leishmaniose/parasitologia , Morfogênese , Psychodidae/parasitologia , Animais , Membrana Celular , Citocinese , Feminino , Flagelos/ultraestrutura , Leishmania/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo
6.
Mem Inst Oswaldo Cruz ; 115: e200113, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33111757

RESUMO

BACKGROUND: Lutzomyia longipalpis-derived cell line (Lulo) has been suggested as a model for studies of interaction between sandflies and Leishmania. OBJECTIVES: Here, we present data of proteomic and gene expression analyses of Lulo cell related to interactions with Leishmania (Viannia) braziliensis. METHODS: Lulo cell protein extracts were analysed through a combination of two-dimensional gel electrophoresis and mass spectrometry and resulting spots were further investigated in silico to identify proteins using Mascot search and, afterwards, resulting sequences were applied for analysis with VectorBase. RESULTS: Sixty-four spots were identified showing similarities to other proteins registered in the databases and could be classified according to their biological function, such as ion-binding proteins (23%), proteases (14%), cytoskeletal proteins (11%) and interactive membrane proteins (9.5%). Effects of interaction with L. (V.) braziliensis with the expression of three genes (enolase, tubulin and vacuolar transport protein) were observed after an eight-hour timeframe and compared to culture without parasites, and demonstrated the impact of parasite interaction with the expression of the following genes: LLOJ000219 (1.69-fold), LLOJ000326 (1.43-fold) and LLOJ006663 (2.41-fold). CONCLUSIONS: This set of results adds relevant information regarding the usefulness of the Lulo cell line for studies with Leishmania parasites that indicate variations of protein expression.


Assuntos
Leishmania braziliensis , Leishmania , Proteômica , Psychodidae , Animais , Linhagem Celular , Leishmania/genética , Leishmania braziliensis/genética , Psychodidae/parasitologia , Transcriptoma
7.
PLoS Negl Trop Dis ; 14(10): e0008748, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33017399

RESUMO

Since leishmaniases are zoonotic vector-borne diseases transmitted through the bites of infected female sand flies, identification of the sources of imbibed blood meals and the detection and identification of leishmanial DNA in them are important in discerning animal reservoirs, clarifying the epidemiology and facilitating control of local leishmaniases. CDC light traps, aspirators and sticky paper traps were used to collect sand flies in four Palestinian foci of both, CL and VL. Phlebotomine species identification was based on morphological keys. Female specimens were screened to detect and identify leishmanial infections, using kDNA-PCR and ITS1-PCR, and engorged female specimens were analyzed to identify the origin of their blood meals, using an RDB blood meal assay based on the amplification of the cytochrome b gene (cytb) of vertebrate mitochondrial DNA (mtDNA). Twenty sand fly species, 11 of the genus Phlebotomus and nine the genus Sergentomyia, were identified. The most abundant species was Ph. papatasi (33.7%), followed by Ph. sergenti (21%). Among the 691 female sand fly specimens, 18.5% (128/691) were positive for leishmanial DNA, using the kDNA-PCR and 6.4% (44/691) were positive using the ITS1-PCR. DNA from parasites of the genus Leishmania was identified in only 1.5% of the infected sand flies. That of Leishmania tropica parasites was detected in six female specimens of Ph. sergenti and that of L. major parasites in two female specimens of Ph. papatasi. Interestingly, two engorged females of the species Se. (Neophlebotomus) sp. were positive for L. tropica DNA. Ninety engorged female sand flies of Ph. papatasi and 104 of Ph. sergenti had fed on a large variety of vertebrate hosts such as humans, hyraxes, rats, cows, goats and birds. Regarding blood-meals showing a mixture from different species of animal host, hyrax and rat blood was revealed in 8/104 (7.7%) females of Ph. sergenti. Detection of hyrax blood in engorged female sand flies of the species Ph. sergenti supports the role of hyraxes being a potential reservoir of L. tropica in Palestinian regions. Rat blood meals might be significant since a few strains L. tropica and L. infantum were isolated from rats. Further studies must be undertaken before conclusions could be drawn.


Assuntos
DNA de Cinetoplasto/análise , Comportamento Alimentar , Leishmania tropica/isolamento & purificação , Psychodidae/fisiologia , Animais , Árabes , DNA de Cinetoplasto/genética , DNA Espaçador Ribossômico/genética , Transmissão de Doença Infecciosa , Feminino , Especificidade de Hospedeiro , Humanos , Insetos Vetores/parasitologia , Leishmania tropica/genética , Leishmaniose/transmissão , Reação em Cadeia da Polimerase , Psychodidae/parasitologia
8.
Sci Rep ; 10(1): 15246, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32943684

RESUMO

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.


Assuntos
Filarioidea/patogenicidade , Insetos Vetores/parasitologia , Leishmania braziliensis , Leishmaniose Cutânea/transmissão , Psychodidae/parasitologia , Animais , Brasil , Complexo IV da Cadeia de Transporte de Elétrons/genética , Feminino , Filarioidea/classificação , Filarioidea/genética , Genes de Helmintos , Genes de Insetos , Humanos , Leishmaniose Cutânea/parasitologia , Masculino , Filogenia , Psychodidae/enzimologia , Psychodidae/genética
9.
Trends Parasitol ; 36(9): 785-795, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32713762

RESUMO

Leishmania parasites have the capacity to rapidly adapt to changing environments in their digenetic life cycle which alternates between a vertebrate and an invertebrate host. Emergence of resistance following drug exposure can evoke phenotypic alterations that affect several aspects of parasite fitness in both hosts. Current studies of the impact of resistance are mostly limited to interactions with the mammalian host and characterization of in vitro parasite growth and differentiation. Development in the vector and transmission capacity have been largely ignored. This review reflects on the impact of drug resistance on its spreading potential with specific focus on the use of the sand fly infection model to evaluate parasite development in the vector and the ensuing transmission potential of drug-resistant phenotypes.


Assuntos
Resistência a Medicamentos , Insetos Vetores/parasitologia , Leishmaniose/transmissão , Psychodidae/parasitologia , Animais , Antiparasitários/farmacologia , Humanos , Leishmania/efeitos dos fármacos , Leishmania/crescimento & desenvolvimento , Leishmaniose/parasitologia , Estágios do Ciclo de Vida/fisiologia
10.
Nat Commun ; 11(1): 3461, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651371

RESUMO

Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa transmitted by infected sand flies. Vaccination through leishmanization with live Leishmania major has been used successfully but is no longer practiced because it resulted in occasional skin lesions. A second generation leishmanization is described here using a CRISPR genome edited L. major strain (LmCen-/-). Notably, LmCen-/- is a genetically engineered centrin gene knock-out mutant strain that is antibiotic resistant marker free and does not have detectable off-target mutations. Mice immunized with LmCen-/- have no visible lesions following challenge with L. major-infected sand flies, while non-immunized animals develop large and progressive lesions with a 2-log fold higher parasite burden. LmCen-/- immunization results in protection and an immune response comparable to leishmanization. LmCen-/- is safe since it is unable to cause disease in immunocompromised mice, induces robust host protection against vector sand fly challenge and because it is marker free, can be advanced to human vaccine trials.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Leishmania major/genética , Leishmania major/patogenicidade , Vacinas Atenuadas/uso terapêutico , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Dexametasona/farmacologia , Feminino , Citometria de Fluxo , Edição de Genes , Engenharia Genética , Humanos , Imunossupressão , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Psychodidae/parasitologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
11.
PLoS One ; 15(6): e0234445, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32579586

RESUMO

This study aimed to describe the sand fly fauna and detect trypanosomatids in these insects from Casa Branca, state of Minas Gerais, Brazil, an endemic area of both visceral (VL) and tegumentary leishmaniasis (TL). Sand flies were collected bimonthly from May 2013 to July 2014, using automatic light traps exposed for three consecutive nights in peridomiciliary areas of nine houses with previous reports of VL and TL. ITS1-PCR and DNA sequencing were performed for trypanosomatids identification. A total of 16,771 sand flies were collected belonging to 23 species. The most abundant species was Nyssomyia whitmani (Antunes & Coutinho, 1939) (70.9%), followed by Lutzomyia longipalpis (Lutz & Neiva, 1912) (15.2%) and Migonemyia migonei (França, 1920) (9.1%). Leishmania amazonensis DNA was detected in Ny. whitmani (four pools) and Le. braziliensis DNA was detected in Psychodopygus lloydi (one pool). In seven pools of Ny. whitmani and in one pool of Lu. longipalpis positive for Leishmania DNA, the parasite species was not determined due to the low quality of the sequences. Moreover, DNA of Herpetomonas spp. was detected in Ny. whitmani (two pools) and Cortelezzii complex (one pool). DNA of Crithidia spp. was detected in Ny. whitmani and Ps. lloydi (both one pool). Our results suggest that Ny. whitmani may be involved in the transmission of Le. amazonensis in the study area. The molecular detection of Le. amazonensis suggests the presence of this species in a sylvatic cycle between vertebrate and invertebrate hosts in the region of Casa Branca. Our data also reveal the occurrence of other non-Leishmania trypanosomatids in sand flies in Casa Branca District.


Assuntos
Biodiversidade , Insetos Vetores/parasitologia , Leishmania/isolamento & purificação , Phlebotomus/parasitologia , Psychodidae/parasitologia , Animais , Brasil/epidemiologia , DNA de Protozoário/isolamento & purificação , Doenças Endêmicas/prevenção & controle , Feminino , Humanos , Leishmania/genética , Leishmaniose/epidemiologia , Leishmaniose/parasitologia , Leishmaniose/prevenção & controle , Leishmaniose/transmissão , Análise de Sequência de DNA
12.
Insect Biochem Mol Biol ; 122: 103393, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32360958

RESUMO

In sandflies, males and females feed on carbohydrates but females must get a blood meal for egg maturation. Using artificial blood meals, this study aimed to understand how galactosamine interferes with sandfly digestive physiology. We also used galactosamine to manipulate the digestive physiology of Lutzomyia longipalpis to investigate its influence on sandfly digestion and Leishmania development within their insect vectors. Galactosamine was capable to reduce Lu. longipalpis trypsinolytic activity in a dose-dependent manner. This effect was specific to galactosamine as other similar sugars were not able to affect sandfly trypsin production. An excess of amino acids supplemented with the blood meal and 15 mM galactosamine was able to abrogate the reduction of the trypsinolytic activity caused by galactosamine, suggesting this phenomenon may be related to an impairment of amino acid detection by sandfly enterocytes. The TOR inhibitor rapamycin reduces trypsin activity in the L. longipalpis midgut. Galactosamine reduces the phosphorylation of the TOR pathway repressor 4EBP, downregulating TOR activity in the gut of L. longipalpis. Galactosamine reduces sandfly oviposition, causes an impact on sandfly longevity and specifically reduces sandfly gut proteases whereas increasing α-glycosidase activity. The administration of 15 and 30 mM galactosamine increased the number of promastigote forms of Le. mexicana and Le. infantum in galactosamine-treated L. longipalpis. Our results showed that galactosamine influences amino acid sensing, reduces sandfly gut protease activity through TOR downregulation, and benefits Leishmania growth within the Lu. longipalpis gut.


Assuntos
Galactosamina/administração & dosagem , Proteínas de Insetos/metabolismo , Leishmania/fisiologia , Peptídeo Hidrolases/metabolismo , Psychodidae/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Animais , Regulação para Baixo , Feminino , Galactosamina/farmacologia , Trato Gastrointestinal/parasitologia , Trato Gastrointestinal/fisiologia , Psychodidae/enzimologia , Psychodidae/parasitologia
13.
Acta Trop ; 209: 105540, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32442434

RESUMO

Earlier research has shown that in vivo immunization with sand fly saliva protects the host against infection by parasites of genus Leishmania, and inoculation of saliva along with Leishmania promastigotes favors infection in the host. In this study, High-Content Imaging System was used to demonstrate in vitro that sand fly saliva also promotes infection by these parasites. THP-1 cells were cultured in 96-well microplates and challenged with three strains of Leishmania braziliensis plus four dilutions of Nyssomyia neivai salivary gland extract. High-Content Imaging System equipment (Operetta CLS, Perkin Elmer) was configured to automatically count both cells and parasites inside the microplates and subsequently calculate the Infection Index (II). Results demonstrate that the extract concentration of 1 gland showed greater infection than other dilutions. These findings suggest that sand fly N. neivai saliva has potential for increasing the parasite infection, reinforcing the importance of studying its components. A new method to evaluate Leishmania infection in vitro assays was also presented, broadening this area of study.


Assuntos
Leishmania braziliensis/crescimento & desenvolvimento , Psychodidae/parasitologia , Saliva/parasitologia , Animais , Feminino , Humanos , Masculino , Camundongos , Células THP-1
15.
Parasit Vectors ; 13(1): 131, 2020 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-32312319

RESUMO

BACKGROUND: The blood-feeding behaviour of female sand flies may increase their likelihood of acquiring and transmitting Leishmania parasites. Studies on the host usage by these insects may thus improve our understanding of the Leishmania transmission risk in leishmaniasis-endemic areas. Here, we developed a fast multiplex real-time PCR assay for simultaneous detection of dog, human and Leishmania DNA in sand flies. METHODS: Primers and TaqMan probes targeting the mitochondrial cytochrome c oxidase subunit 1 and cytochrome b genes of dog and human, respectively, were combined in a multiplex assay, which also includes primers and a TaqMan probe targeting the Leishmania minicircle kinetoplast DNA. RESULTS: The multiplex assay was 100% specific, with analytical sensitivities of 103 fg/reaction for dog and human and 1 fg for Leishmania. By testing field-collected engorged female sand flies (95 Migonemyia migonei and two Nyssomyia intermedia), 50 M. migonei were positive for one or two targets (positivity rates: 45.4% for human, 4.1% for dog and 12.4% for Leishmania DNA). CONCLUSIONS: This multiplex real-time PCR assay represents a novel fast assay for detecting dog, human and Leishmania DNA in female sand flies and therefore a tool for assessing the risk of Leishmania transmission to these hosts in areas of active transmission.


Assuntos
Leishmania/genética , Leishmaniose/diagnóstico , Leishmaniose/veterinária , Reação em Cadeia da Polimerase Multiplex/métodos , Psychodidae/parasitologia , Animais , Ciclo-Oxigenase 1/genética , Primers do DNA/genética , DNA de Cinetoplasto/genética , Cães , Feminino , Humanos , Proteínas de Insetos/genética , Insetos Vetores/parasitologia , Leishmaniose/transmissão , Sensibilidade e Especificidade , Fatores de Tempo
16.
Parasit Vectors ; 13(1): 204, 2020 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-32317026

RESUMO

BACKGROUND: This report describes L. infantum infection seroprevalence in dogs in Spain through data obtained from peer-reviewed literature and a cross-sectional serological survey assessing epidemiological and habitat variables as risk factors for infection. The study also provides preliminary sand fly species distribution data and indicates factors affecting their distribution and density. METHODS: Three different studies were conducted in Spain: (i) a peer-reviewed literature seroprevalence survey (1985-2019); (ii) a cross-sectional serological survey (2011-2016); and (iii) a preliminary entomological survey (2013-2014). In the cross-sectional serological survey, 1739 dogs from 74 different locations including 25 Spanish provinces were tested for L. infantum by indirect immunofluorescence antibody test (IFAT) (antibody titre ≥ 1:100). Seroprevalence of L. infantum infection was analysed by province and bioclimatic zone. Statistics were used to analyse relationships between several dog- and environment-related variables and L. infantum seroprevalence. In parallel, during 2013-2014, sand flies were collected across the Iberian Peninsula and the Balearic Islands using CDC light traps to examine relationships between habitat-related factors and sand fly species densities (number of sand flies per trap per hour). RESULTS: The literature review revealed that the provinces showing the highest seroprevalence were Balearic Islands (57.1%), Ourense (35.6%), Málaga (34.6%) and Cáceres (34.2%), and those showing the lowest seroprevalence were Vizcaya (0%), Cantabria (2.0%) and Álava (3.3%). In our survey, anti-Leishmania IgG antibodies were detected in 176 of the 1739 dogs rendering a seroprevalence of 10.12%. Percentage seroprevalence distributions significantly varied among bioclimatic belts. Seropositivity for L. infantum was related to size (large breed dogs versus small) and were significantly higher in younger dogs (≤ 1 years-old). In the entomological survey, 676 sand flies of five species were captured: 562 (83.13%) Phlebotomus perniciosus; 64 (9.47%) Sergentomyia minuta; 38 (5.62%) P. ariasi: 6 (0.89%) P. sergenti; and 6 (0.89%) P. papatasi. Phlebotomus perniciosus showed a greater density in the thermo-Mediterranean than in the meso-Mediterranean zone. Densities of S. minuta and P. ariasi were significantly higher in rural habitats. CONCLUSIONS: This updated seroprevalence map of L. infantum infection in dogs in Spain defines non-endemic, hypoendemic, endemic and hyperendemic areas, and confirms P. perniciosus as the most abundant sand fly vector in Spain.


Assuntos
Distribuição Animal , Doenças do Cão/epidemiologia , Cães/parasitologia , Leishmaniose Visceral/veterinária , Psychodidae/fisiologia , Animais , Estudos Transversais , Doenças do Cão/parasitologia , Feminino , Insetos Vetores , Leishmania infantum , Leishmaniose Visceral/epidemiologia , Masculino , Psychodidae/parasitologia , Fatores de Risco , Estudos Soroepidemiológicos , Espanha/epidemiologia , Inquéritos e Questionários , Médicos Veterinários
17.
Parasit Vectors ; 13(1): 193, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32293524

RESUMO

BACKGROUND: For long time, canine leishmaniosis (CanL) was considered endemic in the southern, central, and insular regions of Italy, whereas heartworm disease (HW) caused by Dirofilaria immitis was considered endemic in the northern region and in the swampy Po Valley. Following the reports of new foci of both diseases, in this study we update the distribution patterns and occurrence of new foci of CanL and HW discussing the main drivers for the changes in the epidemiology of these two important zoonotic canine vector-borne diseases. METHODS: Based on the statistical analyses of serological assays (n = 90,633) on L. infantum exposure and D. immitis infection performed by two reference diagnostic centres in Italy over a ten-year period (2009-2019) irrespective of the anamnesis of dogs. The distribution patterns of both parasites are herein presented along with the occurrence of new foci. RESULTS: Results highlighted the changing distribution patterns of L. infantum vs D. immitis infection in Italy. CanL is endemic in some areas of northern regions and HW has endemic foci in central and southern regions and islands. Significant differences in L. infantum exposure and HW infection prevalence among the study macroareas were detected. The overall results of the positive tested samples were 28.2% in southern Italy and islands, 29.6% in central Italy and 21.6% in northern Italy for L. infantum and 2.83% in northern Italy, 7.75% in central Italy and 4.97% in southern Italy and islands for HW. HW positivity significantly varied over years (χ2 = 108.401, df = 10, P < 0.0001), gradually increasing from 0.77% in 2009 to 8.47% in 2016-2017. CONCLUSIONS: New potential epidemiological scenarios are discussed according to a range of factors (e.g. environmental modifications, occurrence of competent insect vectors, transportation of infected animals to non-endemic areas, chemoprophylaxis or vector preventative measures), which may affect the current distribution. Overall, the results advocate for epidemiological surveillance programmes, more focussed preventative and control measures even in areas where few or no cases of both diseases have been diagnosed.


Assuntos
Dirofilaria immitis/parasitologia , Dirofilariose/epidemiologia , Doenças do Cão/epidemiologia , Leishmania infantum/parasitologia , Leishmaniose/veterinária , Animais , Dirofilariose/prevenção & controle , Cães , Insetos Vetores/parasitologia , Itália/epidemiologia , Leishmaniose/epidemiologia , Psychodidae/parasitologia , Testes Sorológicos , Zoonoses
18.
PLoS Negl Trop Dis ; 14(4): e0008253, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32324738

RESUMO

BACKGROUND: In the Mediterranean basin, Leishmania infantum is the causative agent of visceral leishmaniasis (VL), a zoonosis in which the dog is the primary domestic reservoir, although wildlife may have a leading role in the sylvatic cycle of the disease in some areas. Infections without disease are very frequent. There is limited information regarding the role that VL patients and asymptomatic infected individuals could be playing in the transmission of L. infantum. Xenodiagnosis of leishmaniasis has been used in this descriptive study to explore the role of symptomatic and asymptomatic infected individuals as reservoirs in a recent focus of leishmaniasis in southwestern Madrid, Spain. METHODOLOGY AND MAIN FINDINGS: Asymptomatic blood donors (n = 24), immunocompetent patients who were untreated (n = 12) or treated (n = 11) for visceral leishmaniasis (VL), and immunocompromised patients with VL (n = 3) were enrolled in the study. Their infectivity to Phlebotomus perniciosus was studied by indirect xenodiagnosis on peripheral blood samples. Quantitative polymerase chain reaction of blood samples from immunocompetent patients untreated for VL and immunocompromised untreated, treated and under secondary prophylaxis for VL was performed. Antibodies against Leishmania were studied by indirect fluorescent antibody and rK39-immunochromatographic tests. A lymphoproliferative assay with a soluble Leishmania antigen was used to screen for leishmaniasis infection in the healthy population. Sixty-two xenodiagnostic tests were carried out and 5,080 sand flies were dissected. Positive xenodiagnosis was recorded in four patients, with different sand fly infection rates: 1 immunosuppressed HIV / L. infantum coinfected asymptomatic patient, 1 immunosuppressed patient with multiple myeloma and symptomatic active VL, and 2 immunocompetent patients with untreated active VL. All blood donors were negative for both xenodiagnosis and conventional PCR. CONCLUSIONS / SIGNIFICANCE: There is no consensus amongst authors on the definition of an 'asymptomatic case' nor on the tools for screening; we, therefore, have adopted one for the sake of clarity. Immunocompetent subjects, both infected asymptomatics and those treated for VL, are limited in number and appear to have no epidemiological relevance. The impact is limited for immunocompetent patients with untreated active VL, whilst immunosuppressed individuals undergoing immunosuppressive therapy and immunosuppressed individuals HIV / L. infantum coinfected were the most infectious towards sand flies. It is noteworthy that the HIV / L. infantum coinfected patient with asymptomatic leishmaniasis was easily infectious to sand flies for a long time, despite being under continuous prophylaxis for leishmaniasis. Accordingly, screening for latent Leishmania infection in HIV-infected patients is recommended in scenarios where transmission occurs. In addition, screening for VL in HIV-infected patients who have spent time in VL-endemic areas should also be implemented in non-endemic areas. More research is needed to better understand if some asymptomatic coinfected individuals contribute to transmission as 'super-spreaders'.


Assuntos
Reservatórios de Doenças , Transmissão de Doença Infecciosa , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/epidemiologia , Leishmaniose Visceral/transmissão , Adulto , Animais , Anticorpos Antiprotozoários/sangue , Doenças Assintomáticas/epidemiologia , DNA de Protozoário/sangue , Feminino , Humanos , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Psychodidae/parasitologia , Espanha/epidemiologia
19.
Acta Trop ; 207: 105492, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32298655

RESUMO

Phlebotomine sand flies are vectors of Leishmania parasites causing different clinical forms of leishmaniases that represent a serious emerging public health problem worldwide. Mexico harbours a large diversity of sand flies, yet only six species have been considered suspected vectors of Leishmania. The disease has been recorded in 25 states, where the State of Veracruz has recorded the highest number of cases with leishmaniases, although no systematic or epidemiological surveillance studies of Leishmania vectors have been carried out in the region. For that reason, the aim of this study was to perform the molecular detection of Leishmania DNA in phlebotomine sand flies collected from a humid tropical region in Veracruz. We confirmed the presence of Leishmania DNA in eight sand fly species. Sand flies with the highest infection were Psathyromyia aclydifera and Pychodopygus panamensis. This is also the first report of Leishmania DNA in Psathyromyia aclydifera, Psathyromyia carpenteri, Dampfomyia beltrani and Brumptomyia mesai. Our findings highlight the importance of entomological surveys and epidemiological studies, since they enable to determine whether sand fly species may be potential Leishmania vectors in a given area of the Mexico, as transmission dynamics can vary in the different regions.


Assuntos
DNA de Protozoário/análise , Leishmania infantum/genética , Psychodidae/parasitologia , Animais , Feminino , Masculino
20.
PLoS Negl Trop Dis ; 14(3): e0008014, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32126078

RESUMO

BACKGROUND: Leishmaniasis is a vector-borne neglected disease. Inside the natural sand fly vector, the promastigote forms of Leishmania undergo a series of extracellular developmental stages to reach the infectious stage, the metacyclic promastigote. There is limited information regarding the expression profile of L. infantum developmental stages inside the sand fly vector, and molecular markers that can distinguish the different parasite stages are lacking. METHODOLOGY/PRINCIPAL FINDINGS: We performed RNAseq on unaltered midguts of the sand fly Lutzomyia longipalpis after infection with L. infantum parasites. RNAseq was carried out at various time points throughout parasite development. Principal component analysis separated the transcripts corresponding to the different Leishmania promastigote stages, the procyclic, nectomonad, leptomonad and metacyclics. Importantly, there were a significant number of differentially expressed genes when comparing the sequential development of the various Leishmania stages in the sand fly. There were 836 differentially expressed (DE) genes between procyclic and long nectomonad promastigotes; 113 DE genes between nectomonad and leptomonad promastigotes; and 302 DE genes between leptomonad and metacyclic promastigotes. Most of the DE genes do not overlap across stages, highlighting the uniqueness of each Leishmania stage. Furthermore, the different stages of Leishmania parasites exhibited specific transcriptional enrichment across chromosomes. Using the transcriptional signatures exhibited by distinct Leishmania stages during their development in the sand fly midgut, we determined the genes predominantly enriched in each stage, identifying multiple potential stage-specific markers for L. infantum. CONCLUSIONS: Overall, these findings demonstrate the transcriptional plasticity of the Leishmania parasite inside the sand fly vector and provide a repertoire of potential stage-specific markers for further development as molecular tools for epidemiological studies.


Assuntos
Expressão Gênica , Marcadores Genéticos , Leishmania infantum/crescimento & desenvolvimento , Leishmania infantum/genética , Psychodidae/parasitologia , Animais , Feminino , Trato Gastrointestinal/parasitologia , Perfilação da Expressão Gênica , Análise de Sequência de RNA
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