Natural infection of Lutzomyia tortura with Leishmania (Viannia) naiffi in an Amazonian area of Ecuador

Natural infection of sand flies with Leishmania parasites was surveyed in an Amazonian area in Ecuador where leishmaniasis is endemic. Seventy-one female sand flies were dissected and one was positive for Leishmania protozoa. The species of this sand fly was identified as Lutzomyia (Lu.) tortura on the basis of morphologic characteristics. Analysis of the cytochrome b gene sequence identified the parasite as L. (Viannia) naiffi. We report the distribution of L. (V.) naiffi in Ecuador and detection of a naturally infected sand fly in the Ecuadorian Amazon and natural infection of Lu. tortura with Leishmania parasites in the New World.     

Monthly variation in natural infection of the sandfly Lutzomyia ayacuchensis with Leishmania mexicana in an endemic focus in the Ecuadorian Andes.

In order to collect information on the role of Lutzomyia ayacuchensis in the transmission of leishmaniasis in a newly discovered Andean endemic focus in Ecuador, a longitudinal field study was carried out over 13 months. Monthly dissections were made of a minimum of 200 anthropophilic sandflies, collected at night during the month. A total of 2600 flies was separated from a small number of Lu. osornoi, another anthropophilic species in the area, and dissected; 95 (3.65%) were naturally infected with Leishmania mexicana promastigotes. The parasites were always located in the sandfly midgut. The current study revealed a marked monthly variation both in natural infections with Leishmania and in biting activity of sandflies in the endemic area, demonstrating a high transmission rate during the period from the early rainy season to the early or mid dry season (February to July).     

Detection and identification of Leishmania species within naturally infected sand flies in the andean areas of ecuador by a polymerase chain reaction

The surveillance of prevalent Leishmania and sand fly species in endemic areas is important for prediction of the risk and expansion of leishmaniasis. In this study, we developed a polymerase chain reaction (PCR)-based method for detection of Leishmania minicircle DNA within individual sand flies. Using this method, we detected minicircle DNA in 6 (3.3%) of 183 sand flies, while 5 (3.5%) of 143 were positive for Leishmania promastigotes in the same areas by microscopic examination. The species were identified as Leishmania (Leishmania) mexicana by nucleotide sequencing of the cytochrome b gene. Additionally, all the Leishmania-positive sand flies were identified as Lutzomyia ayacuchensis by the restriction enzyme digestion of the PCR-amplified 18S ribosomal RNA gene fragments. Since this combined method is relatively easy and can process a large number of samples, it will be a powerful tool for the rapid identification of prevalent sand fly and Leishmania species as well as monitoring the infection rate in sand fly populations in endemic areas.     

Naturally infected Lutzomyia sand flies in a Leishmania-endemic area of Brazil

In Brazil, Leishmania transmission involves several species of phlebotomine sand flies that are closely associated with different parasites and reservoirs, giving rise to different transmission cycles. The present study focused on naturally infected phlebotomines originating from Santa Luzia, a municipality near Belo Horizonte, capital of the Brazilian state of Minas Gerais, in which leishmaniasis are endemic. Systematic and non systematic approaches,involving the use of light traps and direct aspiration from resting sites, respectively, were used to collect females and flies. Identification of the captured insects and determination of natural infection by Leishmania spp. were performed using both conventional dissection methods and polymerase chain reaction (PCR). The dissection of 102 sand flies allowed five species of Lutzomyia to be identified, although no flagellate parasite forms were observed.In addition, 211 sand flies were identified, were separated according to species, and were combined into 11 pools of up to 20 individuals each. PCR analyses showed that two of these pools were infected with Leishmania:one pool of Lu. whitmani was infected with Le. (Viannia) spp. and another of Lu. cortelezzii was infected with Le. chagasi. This suggests that Lu. whitmani may be a possible vector of Leishmania in the study area, and more work needs to be performed to assess the role of Lu. cortelezzii as a vector.     

Establishment of a mass screening method of sand fly vectors for Leishmania infection by molecular biological methods

Surveillance of the prevalence of Leishmania and its vector, sand fly species, in endemic and surrounding areas is important for prediction of the risk and expansion of leishmaniasis. In this study, a method for the mass screening of sand flies for Leishmania infection was established. This method was applied to 319 field-captured specimens, and 5 positive sand flies were detected. Sand fly species were identified by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) of the18S rRNA gene, and all the positive flies were Lu. hartmanni. Furthermore, cytochrome b (Cyt b) gene sequence analyses identified all the parasites as Endotrypanum species including a probable novel species. Because the method requires minimum effort and can process a large number of samples at once, it will be a powerful tool for studying the epidemiology of leishmaniasis.     

Natural infection of Lutzomyia neivai with Leishmania spp. in northwestern Argentina

The natural infection of Lutzomyia neivai with Leishmania in the endemic area of American cutaneous leishmaniasis (ACL) in northwestern Argentina was analyzed by the polymerase chain reaction (PCR)-hybridization technique. Phlebotominae sand flies were captured in the provinces of Tucumán and Salta between 1999 and 2003. From a sample of 440 Lu. neivai females analysed for the detection of the Leishmania (Viannia) and Leishmania (Leishmania) subgenera, 9.1% of the samples resulted infected with a parasite of the subgenus Viannia and none with the Leishmania. This is the first report of naturally infected sand flies in Argentina besides the first report of infected Lu. neivai sensu strictu. Our results contributed to further incrimination of this specie as vector of leishmaniasis in the area and the identification of the main circulating parasite as belonging to the Leishmania (Viannia) subgenera.     

Epidemiology of Andean cutaneous leishmaniasis: incrimination of Lutzomyia ayacuchensis (Diptera: psychodidae) as a vector of Leishmania in geographically isolated, upland valleys of Peru

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

Natural Leishmania Infection of Lutzomyia auraensis in Madre de Dios, Peru, Detected by a Fluorescence Resonance Energy Transfer-Based Real-Time Polymerase Chain Reaction

Abstract. Leishmania species of the Viannia subgenus are responsible for most cases of New World tegumentary leishmaniasis. However, little is known about the vectors involved in disease transmission in the Amazon regions of Peru. We used a novel real-time polymerase chain reaction (PCR) to assess Leishmania infections in phlebotomines collected in rural areas of Madre de Dios, Peru. A total of 1,299 non-blood fed female sand flies from 33 species were captured by using miniature CDC light traps. Lutzomyia auraensis was the most abundant species (63%) in this area. Seven of 164 pools were positive by PCR for Leishmania by kinetoplast DNA. The real-time PCR identified four Lu. auraensis pools as positive for L. (Viannia) lainsoni and L. (V.) braziliensis. The minimum infection prevalence for Lu. auraensis was estimated to be 0.6% (95% confidence interval = 0.20-1.42%). Further studies are needed to assess the importance of Lu. auraensis in the transmission of New World tegumentary leishmaniasis in hyperendemic areas of Peru.     

Leishmaniasis in Colombia. I. Studies on the phlebotomine fauna associated with endemic foci in the Pacific Coast region

Studies on the phlebotomine fauna related to the leishmaniasis endemic foci of the Colombian Pacific Coast were carried out in the municipalities of Tumaco and Buenaventura. In Inguapí del Guadual, Tumaco, Lutzomyia trapidoi and Lu. gomezi were the predominant anthropophilic species; Lu. panamensis and Lu. hartmanni were less frequent. In Bajo Calima, Buenaventura, Lu. trapidoi represented over 94% of the anthropophilic sandflies. Continuous sampling from 1800 to 0600 hours in Inguapí del Guadual demonstrated that Lu. trapidoi bites mainly at dusk and dawn whereas Lu. gomezi remains active throughout the night. In Inguapí del Guadual, promastigotes were found in 0.1% (2/2, 305) of Lu. trapidoi, 0.2% (3/140) of Lu. gomezi, and 0.2% (1/424) of Lu. panamensis samples collected. In Bajo Calima, 1.9% (8/429) of Lu. trapidoi were found to be infected. Leishmania braziliensis panamensis, the most common Leishmania subspecies in the human population of this endemic focus, was isolated from 1 Lu. trapidoi from Inguapí del Guadual. Parasitological and entomological findings suggest that Lu. trapidoi could be the main vector of Leishmania in these areas, although Lu. gomezi and Lu. panamensis were also predominant.     

Current knowledge of Leishmania vectors in Mexico: how geographic distributions of species relate to transmission areas

Leishmaniases are a group of vector-borne diseases with different clinical manifestations caused by parasites transmitted by sand fly vectors. In Mexico, the sand fly Lutzomyia olmeca olmeca is the only vector proven to transmit the parasite Leishmania mexicana to humans, which causes leishmaniasis. Other vector species with potential medical importance have been obtained, but their geographic distributions and relation to transmission areas have never been assessed. We modeled the ecological niches of nine sand fly species and projected niches to estimate potential distributions by using known occurrences, environmental coverages, and the algorithms GARP and Maxent. All vector species were distributed in areas with known recurrent transmission, except for Lu. diabolica, which appeared to be related only to areas of occasional transmission in northern Mexico. The distribution of Lu. o. olmeca does not overlap with all reported cutaneous leishmaniasis cases, suggesting that Lu. cruciata and Lu. shannoni are likely also involved as primary vectors in those areas. Our study provides useful information of potential risk areas of leishmaniasis transmission in Mexico.     

Distribution of Lutzomyia ayacuchensis, the vector of Andean-type cutaneous leishmaniasis,at different altitudes on the Andean slope of Ecuador

Distribution of the vector species is a major risk factor for the endemicity of leishmaniasis. In the present study, the vertical distribution of Lutzomyia (Lu.) ayacuchensis, the vector of Leishmania (Leishmania) mexicana in the Ecuadorian Andes, was surveyed at different altitudes (300–2500 m above sea level) of the Andean slope. The vector species Lu. ayacuchensis was identified at an altitude of 650 m and a higher areas, and higher distribution ratio of the species was observed at higher altitudes. In addition, high ratios of L. (L.) mexicana infection were detected in higher areas, but none in lower populations of sand flies. Since an association between sand fly populations and vector competence is suggested in Lu. ayacuchensis, haplotype analysis was performed on the species from different altitudes of the study areas; however, no apparent difference was observed among populations. These results suggested that Lu. ayacuchensis in Andean slope areas of Ecuador has the potential to transmit L. (L.) mexicana and spread leishmaniasis in these areas.     

不同利什曼原虫分离株在培养基中生长繁殖观察

目的　比较我国不同类型内脏利什曼病流行区利什曼原虫前鞭毛体在不同培养基中的生长繁殖情况,为选择合适培养基用于利什曼原虫培养提供实验依据。方法　将3 ×105个KS?2、Cy、JIASHI?5株利什曼原虫前鞭毛体分别接种至1 mL NNN培养基、1 mL M199 + 20%胎牛血清培养基、1 mL M199 + 20%马血清培养基及1 mL 脑心浸液培养基（含血红素）中,22 ℃温箱中无菌静置培养,显微镜下连续观察计数8 d,绘制3株利什曼原虫前鞭毛体的生长曲线。 结果 KS?2、Cy、JIASHI?5株利什曼原虫前鞭毛体均能在NNN培养基、M199 + 20%胎牛血清培养基和M199 + 20%马血清培养基中生长繁殖,在NNN培养基中培养不同时间后的3株利什曼原虫前鞭毛体计数均显著高于M199 + 20%胎牛血清培养基和M199 + 20%马血清培养基（P均 < 0.05）,在这3种培养基中培养不同时间后的KS?2株利什曼原虫前鞭毛体计数均显著高于Cy和JIASHI?5株（P均 < 0.05）。KS?2、Cy、JIASHI?5株利什曼原虫前鞭毛体均不能在脑心浸液培养基中生长繁殖。结论　分离自我国不同类型内脏利什曼病流行区的利什曼原虫在同一培养基中生长增殖速度有差异,同一利什曼原虫分离株在不同培养基中的生长繁殖速度亦有差异。NNN培养基是最适合我国内脏利什曼病流行区利什曼原虫分离株的培养基。     

不同利什曼原虫分离株在培养基中生长繁殖观察

目的　比较我国不同类型内脏利什曼病流行区利什曼原虫前鞭毛体在不同培养基中的生长繁殖情况，为选择合适培养基用于利什曼原虫培养提供实验依据。方法　将3 ×105个KS?2、Cy、JIASHI?5株利什曼原虫前鞭毛体分别接种至1 mL NNN培养基、1 mL M199 + 20%胎牛血清培养基、1 mL M199 + 20%马血清培养基及1 mL 脑心浸液培养基（含血红素）中，22 ℃温箱中无菌静置培养，显微镜下连续观察计数8 d，绘制3株利什曼原虫前鞭毛体的生长曲线。 结果 KS?2、Cy、JIASHI?5株利什曼原虫前鞭毛体均能在NNN培养基、M199 + 20%胎牛血清培养基和M199 + 20%马血清培养基中生长繁殖，在NNN培养基中培养不同时间后的3株利什曼原虫前鞭毛体计数均显著高于M199 + 20%胎牛血清培养基和M199 + 20%马血清培养基（P均 < 0.05），在这3种培养基中培养不同时间后的KS?2株利什曼原虫前鞭毛体计数均显著高于Cy和JIASHI?5株（P均 < 0.05）。KS?2、Cy、JIASHI?5株利什曼原虫前鞭毛体均不能在脑心浸液培养基中生长繁殖。结论　分离自我国不同类型内脏利什曼病流行区的利什曼原虫在同一培养基中生长增殖速度有差异，同一利什曼原虫分离株在不同培养基中的生长繁殖速度亦有差异。NNN培养基是最适合我国内脏利什曼病流行区利什曼原虫分离株的培养基。     

Detection of natural infection in Lutzomyia cruzi and Lutzomyia forattinii (Diptera: Psychodidae: Phlebotominae) by Leishmania infantum chagasi in an endemic area of visceral leishmaniasis in Brazil using a PCR multiplex assay

In order to identify Lutzomyia spp. naturally infected by Leishmania parasites a PCR multiplex assay coupled to non-isotopic hybridization was used for the analysis of insect samples collected by CDC light traps in an endemic area of visceral leishmaniasis (VL) in the municipality of Corumbá, Mato Grosso do Sul State, Brazil in May/June 2006. Wild sand flies were identified and grouped into pools of 10 female specimens and 27 groups in total were collected. Positive results were obtained from Lutzomyia cruzi (2 out of 13 pools) and Lutzomyia forattinii (1 out of 14 pools). The positive pools were confirmed as being infected by Leishmania infantum chagasi after hybridizing the PCR products with a species-specific biotinylated probe derived from the kinetoplast minicircle conserved sequence. Given that we detected infection in 3 out of 27 groups and that there was at least 1 infected insect in each, it was possible to infer an infection rate of 1.5% for Lu. cruzi and 0.7% for Lu. forattinii in the analyzed samples. These results confirm the vectorial role of Lu. cruzi in transmitting L. infantum chagasi and suggest Lu. forattinii as a potential VL vector in the municipality of Corumbá, where notifications of the disease in humans and dogs have increased over the last two decades.     

新疆克拉玛依皮肤利什曼病传播媒介的研究

１９９４年的研究表明，在克拉玛依从皮肤利什曼病患者皮肤损害部位和从硕大白岭吴氏亚种消化道内分离出来的利什曼原虫，经ＤＮＡ基因型的分析，证实与婴儿利什曼原虫同源。本文报道，在皮肤利什曼病流行区内硕大白蛉吴氏亚种的数最颇大，亲人性强，在野外和居民点内该蛉的前鞭毛体自然感染率分别为５．９％（５８／９８５）和２．９％（１３／４４９），前鞭毛体在该蛉的消化道内能大量繁殖并可移行至咽及喙部；而在非流行区，该蛉的数量很少或无，也未查见前鞭毛体的感染。综合以往和本文的研究结果，作者确认硕大白蛉吴氏亚种为克拉玛依山婴儿利什曼原虫所致的皮肤利什曼病的传播媒介。     

Prevalence of sand flies and Leishmania donovani infection in a natural population of female Phlebotomus argentipes in Bihar State, India

Leishmaniasis is a vector-borne disease, and in the Indian subcontinent the female Phlebotomus argentipes is the vector for Leishmania donovani. However, data on the extent of sand fly infection rates in natural settings using molecular methods have not been extensively reported in India. In this study a PCR technique was applied targeting the 18S rRNA encoding region to determine the prevalence of Leishmania infection in female P. argentipes captured in the field. For this study, sand flies were collected from 897 houses selected from 50 villages endemic for visceral leishmaniasis (VL) in Muzaffarpur district, Bihar state, using CDC miniature light traps and mouth aspirators. A total of 14,585 sand flies were collected of which 449 were female P. argentipes divided into 132 pools. Molecular detection using PCR targeting the 18S rRNA gene was carried out for the identification of P. argentipes and Leishmania. The overall prevalence of infection was 4.90-17.37% for L. donovani in female P. argentipes in endemic regions of Bihar state. In this study no correlation was found between the presence of infected sand flies and the occurrence of clinical VL. This study provides the first report evaluating the prevalence of Leishmania infection in sand flies in a region endemic for VL in India. Sergentomyia species are the most common species of sand fly. Knowledge of the infection rate in female P. argentipes may help in predicting severity of disease and in vector elimination programs.     

Diversity and ecology of sand flies (Diptera: Psychodidae: Phlebotominae) in coastal French Guiana

In French Guiana, at least five Leishmania species are known to be sympatically transmitted in sylvatic ecotopes. However, the previous surveys on the phlebotomine sand fly fauna were published 20 years ago. During that period, many ecological changes have occurred. Sand fly collections were conducted with CDC light traps in five stations representing the main ecotopes of French Guiana. A total of 817 sand flies belonging to 2 genera, 18 sub-genera, and 46 different species were identified. The species Lutzomyia umbratilis (16.6% of the collected specimens), Lu. infraspinosa (12.7%), Lu. ininii (8.0%), and Lu. flaviscutellata (6.1%) were the most common species. The stratification by height, activity period, and resting site preferences of the most abundant sand flies were analyzed. Population abundance and diversity were compared for each ecotope. The potential of certain sand fly species in leishmaniasis transmission is discussed.     

Canine visceral leishmaniasis in Teresina, Brazil: Relationship between clinical features and infectivity for sand flies

Leishmania chagasi is an intracellular parasite transmitted by the bite of the phlebotomine sand fly Lutzomyia longipalpis, which is the most important of American visceral leishmaniasis. In the gut of the vector, amastigoste forms of the parasite transform into metacyclic promastigotes, from there to the foregut, where they could be transmitted in the next blood meal. Xenodiagnosis is an important tool for the detection of Leishmania, especially when associated to molecular techniques, both being useful for the monitoring and evaluation of dog infectivity in endemic areas. In this study, direct search of Leishmania from material obtained through xenodiagnosis performed in dogs captured in Teresina (Piauí State, Brazil) identified that the predominant forms of the parasite were the procyclic and metacyclic forms located in the hindgut, detected between the 5th and 6th day after the blood meal. Using polymerase chain reaction (PCR), we revealed that dogs with different clinical status were able to infect phlebotomines, the rates of sand fly infection being higher for symptomatic dogs (13%) as compared to asymptomatic ones (3.5%). The direct search was able to demonstrate infection only in phlebotomines in which the blood meal was performed on symptomatic dogs, with a rate of infection of 1.6%. The results underline the importance of using PCR and xenodiagnosis for the detection of Leishmania sp. And for the evaluation of infectivity of dogs in endemic areas, especially those that are asymptomatic.     

Domestic swine in a visceral leishmaniasis endemic area produce antibodies against multiple Leishmania infantum antigens but apparently resist to L. infantum infection

In order to investigate whether pigs can be infected by Leishmania infantum, a serological and parasitological study was carried out on swine in the Jequié municipality, Northeast of Brazil. Anti-Leishmania infantum antibodies were detected in 37 out of 92 swine (40.2%), by two different assays: an anti-L. infantum lysate and an anti-K39 recombinant protein ELISA. An experimental study was also carried out to verify the susceptibility of domestic pigs to L. infantum infection. Three sows inoculated with 10(8) stationary-phase infective L. infantum promastigotes (26% metacyclic promastigotes) per kilogram of body weight produced anti-Leishmania antibodies until the end of the experiment, 11 months later. No parasites, however, could be visualized through optical microscopy of spleen, liver and bone marrow or by in vitro culture of these organs. Homogenates of these organs were also inoculated in hamsters, without producing infection. No Leishmania DNA was detected by polymerase chain reaction (PCR) in sand flies fed on these animals. The results indicate that domestic pigs bitten by L. infantum-infected vectors in the endemic area do not display a full infection pattern, and the positive association in endemic areas between the presence of swine and infection in canines may not be ascribable to the former acting as a parasite reservoir.     

Ecology of sand flies in a low-density residential rural area,with mixed forest/agricultural exploitation,in north-eastern Brazil

Cutaneous leishmaniasis caused by Leishmania braziliensis is endemic in Brazil, where Lutzomyia whitmani is the most important vector involved in the transmission to humans, particularly in the peridomestic environment. Herein, we assessed the ecology of sand flies, including Lu. whitmani, in a low-density residential rural area with mixed forest/agricultural exploitation in north-eastern Brazil, where cutaneous leishmaniasis is endemic. Particularly, we hypothesized that sand fly abundance was correlated with climatic variables. Sand fly collections were carried out monthly from August 2013 to August 2014, using seven CDC light traps, for three consecutive nights, in three kinds of environments: indoor, peridomicile and forest. Collected sand flies were identified based on morphology and females of Lu. whitmani (n = 169), Lu. amazonensis (n = 134) and Lu. complexa (n = 21) were selected and tested by PCR for Leishmania (Viannia) spp. In total, 5167 sand flies belonging to 19 species were identified, being that Lu. choti (43.2%) was the most frequent species, followed by Lu. amazonensis (16.6%), Lu. whitmani (15.8%), Lu. sordellii (10.7%) and Lu. quinquefer (5.8%), which together represented over 90% of the collected sand flies. All females tested by PCR were negative. The number of sand flies collected daily was positively correlated with temperature and negatively correlated with rainfall and relative humidity. Furthermore, there was a positive correlation between daily number of sand flies and daily average saturation deficit. This study points out that the number of sand flies captured daily is correlated to climatic variables, including saturation deficit, which may represent a useful parameter for monitoring sand fly populations in leishmaniasis-endemic areas.