RESUMO
Leishmania (V.) braziliensis, the causative agent of mucocutaneous leishmaniasis in the New World, may present an LD1 type genomic amplification that appears as a small 245 kb linear chromosome, and is not clearly associated to the presence of a selection agent. A bt1 gene, codifying for a biopterin transporter protein, was identified in this small chromosome. Leishmania are auxotrophic for pterins and one of the proposed explanations for the appearance of this amplification is the improvement of biopterin capture by the parasite. We analyzed some biological aspects of two lineages of L. braziliensis strain M2903, with and without the small amplified chromosome. We showed differences in infectivity of these lineages, in macrophages and the insect vector Lutzomyia longipalpis, as well as in the uptake and metabolization of intermediates of the Leishmania biopterin salvage pathway. Our results suggest that the genomic amplification favors survival due to improved biopterin capture and at the same time hinders the infective capability, suggesting that within a population different parasites can perform different roles.
Assuntos
Leishmania braziliensis/genética , Leishmania braziliensis/patogenicidade , Leishmaniose Mucocutânea/parasitologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Protozoários/genética , Animais , Linhagem Celular , Cromossomos/genética , Amplificação de Genes , Insetos Vetores/parasitologia , Leishmania braziliensis/metabolismo , Macrófagos/parasitologia , Metotrexato/farmacologia , Camundongos , Pteridinas/metabolismoRESUMO
Lutzomyia longipalpis is the principal vector of visceral leishmaniasis in the Americas, and can also transmit some viruses. To help develop a gene-silencing system for this sandfly, we transfected cultured embryonic cells with various double-stranded RNAs using West Nile virus (WNV) virus-like particles (VLPs) expressing luciferase as the target RNA to demonstrate effective gene knock-down. When luciferase dsRNA was introduced into these cells, they produced the expected reduction in VLP-encoded luciferase, suggesting specific silencing of the luciferase gene. Surprisingly, we found that unrelated dsRNAs, which included those specific for several L. longipalpis gene sequences and Escherichia coli beta-galactosidase, diminished replication of the VLP-encoded genome. These results are the first indication for a nucleic acid-induced, non-specific antiviral response in this important insect vector.
Assuntos
Luciferases/genética , Psychodidae/genética , RNA de Cadeia Dupla/genética , Vírus do Nilo Ocidental/genética , Aedes , Animais , Células Cultivadas , Expressão Gênica , Luciferases/metabolismo , Psychodidae/citologia , Psychodidae/virologia , RNA Interferente Pequeno/genética , Transfecção , Replicação Viral/genética , Vírus do Nilo Ocidental/crescimento & desenvolvimento , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Visceral leishmaniasis (VL) is a serious tropical disease that affects approximately 500 thousand people worldwide every year. In the Americas, VL is caused by the parasite Leishmania (Leishmania) infantum chagasi mainly transmitted by the bite of the sand fly vector Lutzomyia longipalpis. Despite recent advances in the study of interaction between Leishmania and sand flies, very little is known about sand fly protein expression profiles. Understanding how the expression of proteins may be affected by blood feeding and/or presence of parasite in the vector's midgut might allow us to devise new strategies for controlling the spread of leishmaniasis. In this work, we report the characterization of a vacuolar ATPase subunit C from L. longipalpis by screening of a midgut cDNA library with a 220 bp fragment identified by means of differential display reverse transcriptase-polymerase chain reaction analysis. The expression of the gene varies along insect development and is upregulated in males and bloodfed L. longipalpis, compared to unfed flies.
Assuntos
Comportamento Alimentar/fisiologia , Insetos Vetores/genética , Psychodidae/genética , ATPases Vacuolares Próton-Translocadoras/genética , Animais , Sequência de Bases , Southern Blotting , Clonagem Molecular , Cricetinae , Sistema Digestório/enzimologia , Sistema Digestório/parasitologia , Insetos Vetores/embriologia , Insetos Vetores/enzimologia , Leishmaniose Visceral/transmissão , Masculino , Dados de Sequência Molecular , Subunidades Proteicas , Psychodidae/embriologia , Psychodidae/enzimologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Visceral leishmaniasis (VL) is a serious tropical disease that affects approximately 500 thousand people worldwide every year. In the Americas, VL is caused by the parasite Leishmania (Leishmania) infantum chagasi mainly transmitted by the bite of the sand fly vector Lutzomyia longipalpis. Despite recent advances in the study of interaction between Leishmania and sand flies, very little is known about sand fly protein expression profiles. Understanding how the expression of proteins may be affected by blood feeding and/or presence of parasite in the vector's midgut might allow us to devise new strategies for controlling the spread of leishmaniasis. In this work, we report the characterization of a vacuolar ATPase subunit C from L. longipalpis by screening of a midgut cDNA library with a 220 bp fragment identified by means of differential display reverse transcriptase-polymerase chain reaction analysis. The expression of the gene varies along insect development and is upregulated in males and bloodfed L. longipalpis, compared to unfed flies.
Assuntos
Animais , Masculino , Cricetinae , Comportamento Alimentar/fisiologia , Insetos Vetores/genética , Psychodidae/genética , ATPases Vacuolares Próton-Translocadoras/genética , Sequência de Bases , Southern Blotting , Clonagem Molecular , Sistema Digestório/enzimologia , Sistema Digestório/parasitologia , Insetos Vetores/embriologia , Insetos Vetores/enzimologia , Leishmaniose Visceral/transmissão , Dados de Sequência Molecular , Subunidades Proteicas , Psychodidae/embriologia , Psychodidae/enzimologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
The immunization of vertebrate hosts with vector components may be an alternative for the control of diseases transmitted by insects. In the present study we evaluated the effects of anti-sandfly antibodies on some of the biological parameters of female Lutzomyia longipalpis, a vector of visceral leishmaniasis. Rabbits were immunized with extracts of gut from blood-fed (GB) or sugar-fed (GS) females, carcass of sugar-fed (CS) or blood-fed (CB) females, and with repeated sandfly bites (BITE). Immune sera showed increased antibody titers compared to pre-immunized animals, and specific bands were detected by Western Blot. An analysis of biological parameters revealed a decline in fecundity in the group of females fed on rabbits immunized with GB and BITE. Longevity and mortality were studied in females with oviposition (parous) and without oviposition (nulliparous). Nulliparous females that fed on rabbits immunized with bites died in the highest percentage. A mortality analysis after egg laying revealed a peak on the fifth day in all the groups, but females fed on rabbit subjected to repeated bites showed a shift towards the third day.
Assuntos
Anticorpos/imunologia , Soros Imunes/farmacologia , Insetos Vetores/imunologia , Psychodidae/imunologia , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Feminino , Fertilidade , Longevidade , CoelhosRESUMO
A imunização de hospedeiros vertebrados com componentes derivados de vetores pode se constituir numa estratégia alternativa para o controle de doenças transmitidas por insetos. No presente estudo avaliamos o efeito de anticorpos antiflebótomos sobre alguns parâmetros biológicos de fêmeas de Lutzomyia longipalpis, vetor de leishmaniose visceral. Coelhos foram imunizados com extratos de tubos digestivos de fêmeas alimentadas com açúcar (GS), fêmeas alimentadas com sangue (GB), carcaças de fêmeas alimentadas com açúcar (CS) ou carcaças de fêmeas alimentadas com sangue (CB), e coelho imunizado por repetidas picadas de fêmeas de flebótomos (BITE). Os soros imunes de coelhos apresentaram títulos aumentados quando comparados com os soros pré-imunes, e bandas específicas foram detectadas por meio de Western Blot. A análise dos parâmetros biológicos revelou um decréscimo na fecundidade no grupo de fêmeas alimentadas em coelho imunizado com GB e BITE. A longevidade e a mortalidade foram estudadas em fêmeas com postura (paridas) e fêmeas sem postura (nulíparas). Fêmeas nulíparas que se alimentaram em coelho imunizado por repetidas picadas morreram em maior percentual. A análise da mortalidade, após a postura dos ovos, revelou um pico no quinto dia em todos os grupos, mas em fêmeas que se alimentaram em coelho submetido a repetidas picadas, foi antecipada para o terceiro dia.
Assuntos
Coelhos , Animais , Feminino , Anticorpos/imunologia , Soros Imunes/farmacologia , Insetos Vetores/imunologia , Psychodidae/imunologia , Western Blotting , Ensaio de Imunoadsorção Enzimática , Fertilidade , LongevidadeRESUMO
Trypanosoma vivax and Trypanosoma evansi are livestock parasites of economic importance in Africa, Asia and South America. In the Pantanal, Brazil, they cause economic losses in both cattle and equines. Little is known of their maintenance and spread in nature, particularly in terms of reservoirs and means of mechanical transmission. Here we report for the first time the use of PCR for the detection of T. vivax and T. evansi in bovines, buffaloes and sheep. Whereas parasitological diagnosis detected only two T. vivax infections, one in buffalo and another in a cow, PCR detected infections in 34.8% buffaloes, 44.7% bovines and 37.3% sheep. Trypanozoon primers detected 41.8% infections in buffaloes and 8.1% in cattle. PCR revealed 6.9% mixed infections in buffaloes and 5.3% in cattle. The potential role of cattle and buffaloes as hosts and reservoirs of T. vivax is discussed, as well as the implications of possible extravascular foci in the maintenance of livestock trypanosomosis.
Assuntos
Búfalos/parasitologia , Reação em Cadeia da Polimerase/veterinária , Doenças dos Ovinos/epidemiologia , Trypanosoma/isolamento & purificação , Tripanossomíase/veterinária , Animais , Brasil/epidemiologia , Bovinos , DNA de Protozoário/análise , Reservatórios de Doenças/veterinária , Reação em Cadeia da Polimerase/métodos , Ovinos , Doenças dos Ovinos/diagnóstico , Trypanosoma/genética , Trypanosoma vivax/genética , Trypanosoma vivax/isolamento & purificação , Tripanossomíase/diagnóstico , Tripanossomíase/epidemiologia , Tripanossomíase Bovina/diagnóstico , Tripanossomíase Bovina/epidemiologiaRESUMO
Leishmania (V.) braziliensis M2903 presents a small linear and stable 245 kb chromosome originating from a genomic amplification. Similar amplifications present in other species of Leishmania contain a gene coding for a biopterin transporter. Since Leishmania is auxotrophic for this metabolite, this amplification could result from the need to better capture biotpterin from growth media under specific circumstances. In this paper we show that this gene is also present in L. (V.) braziliensis small chromosome, which shares sequences with other genomic amplifications already described.
Assuntos
Biopterinas/genética , Leishmania braziliensis/genética , Animais , Cromossomos , Eletroforese em Gel de Campo Pulsado , Amplificação de Genes , Genes de Protozoários/genéticaRESUMO
Leishmania (V.) braziliensis M2903 presents a small linear and stable 245 kb chromosome originating from a genomic amplification. Similar amplifications present in other species of Leishmania contain a gene coding for a biopterin transporter. Since Leishmania is auxotrophic for this metabolite, this amplification could result from the need to better capture biotpterin from growth media under specific circumstances. In this paper we show that this gene is also present in L. (V.) braziliensis small chromosome, which shares sequences with other genomic amplifications already described
Assuntos
Animais , Biopterinas , Leishmania braziliensis , Cromossomos , Eletroforese em Gel de Campo Pulsado , Amplificação de Genes , Genes de ProtozoáriosRESUMO
During development within the midgut of the sand fly vector, Leishmania parasites after undergoing differentiation and multiplication must escape the peritrophic matrix (PM). Although Leishmania chitinase is believed to take part in promoting the escape of the parasite from the PM by inducing degradation of chitin fibers, it is conceivable that a sand fly-derived chitinase can also have a role in such an event. Here we describe the molecular cloning and partial characterization of a complete cDNA from a putative gut-specific, blood-induced chitinase from the sand fly vector Lutzomyia longipalpis. Llchit1 has an ORF of 1425 bp that encodes a predicted 51.6 kDa mature protein showing high similarity with chitinases from several different organisms. Messenger RNA expression studies indicate that Llchit1 is detected only in the blood fed midgut and it seems to reach a peak at approximately 72 h post blood meal (PBM). To date, only one midgut-specific chitinase from an insect disease vector, AgChi-1 from Anopheles gambiae, has been characterized. As with its mosquito counterpart, Llchit1 can be a target for development of a transmission blocking vaccine.