ABSTRACT
Immunization with various Leishmania species lacking centrin induces robust immunity against a homologous and heterologous virulent challenge, making centrin mutants a putative candidate for a leishmaniasis vaccine. Centrin is a calcium-binding cytoskeletal protein involved in centrosome duplication in higher eukaryotes and Leishmania spp. lacking centrin are unable to replicate in vivo and are non-pathogenic. We developed a centrin-deficient Leishmania braziliensis (LbCen-/-) cell line and confirmed its impaired survival following phagocytosis by macrophages. Upon experimental inoculation into BALB/c mice, LbCen-/- failed to induce lesions and parasites were rapidly eliminated. The immune response following inoculation with LbCen-/- was characterized by a mixed IFN-γ, IL-4, and IL-10 response and did not confer protection against L. braziliensis infection, distinct from L. major, L. donovani, and L mexicana centrin-deficient mutants. A prime-boost strategy also did not lead to a protective immune response against homologous challenge. On the contrary, immunization with centrin-deficient L. donovani (LdonCen-/-) cross-protected against L. braziliensis challenge, illustrating the ability of LdonCen-/- to induce the Th1-dominant protective immunity needed for leishmaniasis control. In conclusion, while centrin deficiency in L. braziliensis causes attenuation of virulence, and disrupts the ability to cause disease, it fails to stimulate a protective immune response.
ABSTRACT
Leishmania braziliensis is the main causative agent of Tegumentary Leishmaniasis in the Americas. However, difficulties related to genome manipulation, experimental infection, and parasite growth have so far limited studies with this species. CRISPR-Cas9-based technology has made genome editing more accessible, and here we have successfully employed the LeishGEdit approach to attenuate L. braziliensis. We generated a transgenic cell line expressing Cas9 and T7 RNA polymerase, which was employed for the targeted deletion of centrin, a calcium-binding cytoskeletal protein involved in the centrosome duplication in eukaryotes. Centrin-deficient Leishmania exhibit growth arrest at the amastigote stage. Whole-genome sequencing of centrin-deficient L. braziliensis (LbCen-/- ) did not indicate the presence of off-target mutations. In vitro, the growth rates of LbCen-/- and wild-type promastigotes were similar, but axenic and intracellular LbCen-/- amastigotes showed a multinucleated phenotype with impaired survival following macrophage infection. Upon inoculation into BALB/c mice, LbCen-/- were detected at an early time point but failed to induce lesion formation, contrary to control animals, infected with wild-type L. braziliensis. A significantly lower parasite burden was also observed in mice inoculated with LbCen-/- , differently from control mice. Given that centrin-deficient Leishmania sp. have become candidates for vaccine development, we propose that LbCen-/- can be further explored for the purposes of immunoprophylaxis against American Tegumentary Leishmaniasis.
Subject(s)
Leishmania braziliensis , Leishmania , Leishmaniasis, Cutaneous , Animals , CRISPR-Cas Systems , Leishmania braziliensis/genetics , Leishmaniasis, Cutaneous/parasitology , Mice , Mice, Inbred BALB C , Trimethoprim, Sulfamethoxazole Drug CombinationABSTRACT
INTRODUCTION: The classification of the cuticular extensions of insects has been proposed as a taxonomic tool; however, the internal and external processes of the cuticular extensions of the Triatominae subfamily have not been fully analyzed and categorized. Objective: To describe the setae from different regions of the outer cuticle of several triatomine species by scanning electron microscopy. Materials and methods: Triatomines were washed and dried, after which different regions of the body from Eratyrus mucronatus, Triatoma maculata, Panstrongylus geniculatus and Belminus ferroae specimens were dissected, mounted on graphite double-sided adhesive tape over metal supports, metalized with gold and micrographed for further analysis. Results: We described nine types of cuticular setae. We found five types of setae in B. ferroae and T. maculata, four in P. geniculatus and only one in E. mucronatus. According to the proposed typology, type 3 seta was the most common in T. maculata, P. geniculatus and E. mucronatus, whereas type 1a predominated in B. ferroae. Conclusion: Type 3 seta was the most common in the Triatomini tribe (T. maculata, E mucronatus and P. geniculatus), whereas type 1a seta was specific to B. ferroae (tribe Bolboderini), suggesting that the surface morphology of the setae may have taxonomic value at tribe taxonomic level.
Subject(s)
Triatominae/anatomy & histology , Abdomen , Animals , Genitalia , Panstrongylus , SensillaABSTRACT
Introducción. Se han propuesto clasificaciones de las extensiones cuticulares de los insectos para utilizarlas como herramienta taxonómica. A pesar de los diversos estudios morfológicos adelantados en triatominos, el conocimiento que se tiene sobre la distribución y los tipos morfológicos de estas estructuras en la subfamilia aún es escaso y fragmentario. Objetivo. Describir las setas de diferentes regiones de la cutícula externa de varias especies de triatominos mediante microscopía electrónica de barrido. Materiales y métodos. Se lavaron y se secaron especímenes de Eratyrus mucronatus, Triatoma maculata, Panstrongylus geniculatus y Belminus ferroae, y a continuación se disecaron diferentes regiones de sus cuerpos y se montaron sobre cinta adhesiva de grafito de doble faz en soportes metálicos. Se metalizaron con oro y se tomaron las micrografías para su posterior análisis. Resultados. Se describieron nueve tipos de setas cuticulares. En B. ferroae y T. maculata se encontraron cinco tipos de setas, en P. geniculatus, cuatro, y en E. mucronatus, un solo tipo. Según la tipología propuesta, la seta de tipo 3 fue la más común en cada especie, excepto en B. ferroae, en la cual predominó la seta de tipo 1a. Conclusión. La seta más común en las especies de la tribu Triatomini (T. maculata, E. mucronatus y P. geniculatus) fue la de tipo 3; la seta de tipo 1a fue específica para la especie B. ferroae, taxón de la tribu Bolboderini, lo cual permite suponer que la morfología superficial de las setas podría tener valor taxonómico a nivel de tribu.
Introduction: The classification of the cuticular extensions of insects has been proposed as a taxonomic tool; however, the internal and external processes of the cuticular extensions of the Triatominae subfamily have not been fully analyzed and categorized. Objective: To describe the setae from different regions of the outer cuticle of several triatomine species by scanning electron microscopy. Materials and methods: Triatomines were washed and dried, after which different regions of the body from Eratyrus mucronatus, Triatoma maculata, Panstrongylus geniculatus and Belminus ferroae specimens were dissected, mounted on graphite double-sided adhesive tape over metal supports, metalized with gold and micrographed for further analysis. Results: We described nine types of cuticular setae. We found five types of setae in B. ferroae and T. maculata, four in P. geniculatus and only one in E. mucronatus. According to the proposed typology, type 3 seta was the most common in T. maculata, P. geniculatus and E. mucronatus, whereas type 1a predominated in B. ferroae. Conclusion: Type 3 seta was the most common in the Triatomini tribe (T. maculata, E mucronatus and P. geniculatus), whereas type 1a seta was specific to B. ferroae (tribe Bolboderini), suggesting that the surface morphology of the setae may have taxonomic value at tribe taxonomic level.
Subject(s)
Triatominae , Microscopy, Electron, Scanning , SensillaABSTRACT
INTRODUCTION: In recent years, the assassin bug, Panstrongylus geniculatus, has been found infected with Trypanosoma cruzi in rural and urban areas of Caracas, Venezuela. Although historically this insect has been considered a forest species, it has become adapted to more urban artificial environments. OBJECTIVE: The presence of sexual dimorphism was determined as an indicator of adaptation to domiciles. MATERIAL AND METHODS: By Generalized Procrustes Analysis (GPA) and Elliptical Fourier Analysis (EFA), the isometric size and shape of wings, head and pronotums of P. geniculatus was assessed for actively and passively captured specimens. These were collected within domiciles in urban areas of Petare and Altagracia in Caracas City, and from rural or wild environments of Sanare in Andres Eloy Blanco in the state of Lara. RESULTS: Sexual dimorphism was observed in the Sanare specimens, with female wings consistently larger than male wings. Similarly, female wings and heads from bugs captured in Caracas were smaller than those of female bugs captured in Sanare. No significative differences in the conformation of the pronotum were found between male and female bugs. CONCLUSIONS: Based on the assumption that the sexual dimorphism of bugs is reflected by smaller size in domesticated triatomines than in wild bugs, the conclusion is that Caracas P. geniculatus has become adapted to living indoors. This represents an additional risk factor for the Chagas disease transmission in Caracas.
Subject(s)
Insect Vectors/anatomy & histology , Panstrongylus/anatomy & histology , Adaptation, Physiological , Animals , Chagas Disease/transmission , Ecosystem , Female , Humans , Male , Rural Population , Sex Characteristics , Urban Population , VenezuelaABSTRACT
INTRODUCTION: Immature and adult forms of Triatoma maculata have been captured repeatedly in and around the homes in the town of Xaguas, Venezuela. Because of its potential as a Chagas disease vector, a study was undertaken to evaluate the effect of human blood feeding on the viability and spatial structuring of a laboratory colony of this species. OBJECTIVE: The effect of human blood feeding was determined for the viability of a T. maculata laboratory colony, as well as its spatial structuring. MATERIAL AND METHODS: Insects were fed with human blood on artificial feeder. Spatial structuring was undertaken by the generalized analysis of by geometric morphometry. RESULTS: The average fecundity of 27.7 eggs/female/lifetime was found, with a mean time to oviposition of 32.7 days, and a female longevity of 39.2 days. The longest inter-molt period was at the fifth nymphal stage (45.9 days), whereas the shortest was at 18.4 days, during the first nymphal stage. The highest mortality of nymphs was observed at the fifth nymphal stage (77.8%). The lowest molting percentage was observed in the fifth nymphal stage (22.2%). No differences in the size of wings and heads were detectable; although differences in the head shape of individuals of the same sex from different environments were noted. Wing-shape differences were found only between the males of peridomestic and domestic ecotopes. CONCLUSIONS: Triatoma maculata may be entering human dwellings to feed on non-human animals, or alternatively, may be in an incipient state of adaptation to a domestic ecotope for feeding on human beings.
Subject(s)
Blood , Triatoma/physiology , Animals , Animals, Laboratory , Entomology/methods , Humans , Triatoma/anatomy & histology , Triatoma/growth & developmentABSTRACT
Introducción. En años recientes se han encontrado P. geniculatus infectados con Trypanosoma cruzi en ambientes rurales y urbanos de Caracas, Venezuela, lo que indica adaptaciones de dicha especie, considerada históricamente como silvestre, a ambientes artificiales. Objetivo. Determinar la presencia de dimorfismo sexual como indicador de adaptación al domicilio. Materiales y métodos. Mediante análisis generalizado de Procrustes y análisis elíptico de Fourier, se analizaron el tamaño isométrico y la conformación de alas, cabezas y pronotos de P. geniculatus capturados activa y pasivamente en domicilios en sectores urbanos de Altagracia y Petare de Caracas, y provenientes de ambientes rurales o silvestres de Sanare en el municipio Andrés Eloy Blanco en el estado Lara. Resultados. Se observó dimorfismo sexual al considerar el tamaño de las alas en los especímenes capturados en Sanare, las cuales fueron mayores en las hembras que los machos. Asimismo, las alas y cabezas de las hembras capturadas en Caracas fueron más pequeñas comparadas con las de las hembras provenientes de Sanare. No se encontraron diferencias significativas en cuanto a la conformación del pronoto. Conclusiones. Con base en el supuesto de que, por dimorfismo sexual, el tamaño es más reducido en triatominos domiciliados que en los silvestres, se concluye que P. geniculatus de Caracas están adaptados al domicilio, lo que constituye un factor de riesgo en la transmisión de la enfermedad de Chagas en dicha ciudad.
Introduction. In recent years, the assassin bug, Panstrongylus geniculatus, has been found infected with Trypanosoma cruzi in rural and urban areas of Caracas, Venezuela. Although historically this insect has been considered a forest species, it has become adapted to more urban artificial environments. Objective. The presence of sexual dimorphism was determined as an indicator of adaptation to domicilies. Material and methods. By Generalized Procrustes Analysis (GPA) and Elliptical Fourier Analysis (EFA), the isometric size and shape of wings, head and pronotums of P. geniculatus was assessed for actively and passively captured specimens. These were collected within domiciles in urban areas of Petare and Altagracia in Caracas City, and from rural or wild environments of Sanare in Andres Eloy Blanco in the state of Lara. Results. Sexual dimorphism was observed in the Sanare specimens, with female wings consistently larger than male wings. Similarly, female wings and heads from bugs captured in Caracas were smaller than those of female bugs captured in Sanare. No significative differences in the conformation of the pronotum were found between male and female bugs. Conclusions. Based on the assumption that the sexual dimorphism of bugs is reflected by smaller size in domesticated triatomines than in wild bugs, the conclusion is that Caracas P. geniculatus has become adapted to living indoors. This represents an additional risk factor for the Chagas disease transmission in Caracas.
Subject(s)
Body Weights and Measures , Chagas Disease , Triatominae , Sex CharacteristicsABSTRACT
Introducción. Dado el reiterado hallazgo de adultos y formas inmaduras de Triatoma maculata capturados en domicilios y peridomicilios en la localidad de Xaguas (Venezuela), se analizó la viabilidad de una colonia de laboratorio de esta especie alimentada con sangre humana y su estructura espacial. Objetivos. Analizar la viabilidad del ciclo de T. maculata alimentado con sangre humana en condiciones de laboratorio y caracterizar la estructura espacial de una población de T. maculata proveniente de ambientes domiciliarios y peridomiciliarios del centro-occidente de Venezuela. Materiales y métodos. La alimentación con sangre humana se llevó a cabo en lotes por estadio, utilizando un alimentador artificial, y la estructura espacial de los insectos por sexo y ambiente fue analizada mediante morfometría geométrica. Resultados. Los hallazgos fueron: fecundidad promedio, 27,70 huevos/hembra/vida, tiempo promedio de postura de huevos, 32,74 días, y longevidad promedio de la hembra, 39,15 días; el mayor tiempo en V estadio fue de 45,9 días y el menor en I estadio, 18,41 días; la mayor mortalidad fue de 77,78% y el menor porcentaje de muda en V estadio, de 22,23%.No se encontraron diferencias en el tamaño de alas y cabezas; sí se encontraron diferencias de conformación entre las cabezas de un mismo sexo de diferentes hábitats; sólo se encontraron diferencias de conformación de alas entre los machos del domicilio y peridomicilio. Conclusiones. T. maculata de la localidad de Xaguas podría encontrarse en un estado de adaptación al domicilio con fuentes sanguíneas diferentes al humano y en un estado incipiente de adaptación al domicilio con la sangre humana como fuente de alimentación.
Introduction. Immature and adult forms of Triatoma maculata have been captured repeatedly in and around the homes in the town of Xaguas, Venezuela. Because of its potential as a Chagas disease vector, a study was undertaken to evaluate the effect of human blood feeding on the viability and spatial structuring of a laboratory colony of this species. Objective. The effect of human blood feeding was determined for the viability of a T. maculata laboratory colony, as well as its spatial structuring. Material and methods. Insects were fed with human blood on artificial feeder. Spatial structuring was undertaken by the generalized analysis of by geometric morphometry. Results. The average fecundity of 27.7 eggs/female/lifetime was found, with a mean time to oviposition of 32.7 days, and a female longevity of 39.2 days. The longest inter-molt period was at the fifth nymphal stage (45.9 days), whereas the shortest was at 18.4 days, during the first nymphal stage. The highest mortality of nymphs was observed at the fifth nymphal stage (77.8%). The lowest molting percentage was observed in the fifth nymphal stage (22.2%). No differences in the size of wings and heads were detectable; although differences in the head shape of individuals of the same sex from different environments were noted. Wing-shape differences were found only between the males of peridomestic and domestic ecotopes.